Compounds, compositions, methods, and kits relating to telomere extension

ABSTRACT

Compounds and compositions for the transient expression of exogenous telomerase activity in a cell are provided. The compounds and compositions, which relate to a ribonucleic acid coding for a telomerase reverse transcriptase, are useful in the extension of telomeres in cells needing such treatment. Such cells include, for example, cells that contain shortened telomeres and cells from subjects that may benefit from telomere extension, for example subjects that suffer from, or are at risk of suffering from, age-related or other illnesses. Also provided are methods of extending telomeres through the administration of the provided compounds and compositions to animal cells, either in vitro or in vivo, and kits including the compounds or compositions and instructions for use.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/768,047, filed on Feb. 22, 2013, the disclosure of which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under contract AR063963awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Telomeres comprise repetitive DNA sequences at the ends of linearchromosomes that, when sufficiently long, allow each chromosome end toform a loop that protects the ends from acting as double-stranded orsingle-stranded DNA breaks. Artandi & DePinho (2010) Carcinogenesis31:9-18. Telomeres shorten over time, due in part to oxidative damageand incomplete DNA replication, eventually leading to critically shorttelomeres unable to form the protective loop, exposure of the chromosomeends, chromosome-chromosome fusions, DNA damage responses, and cellularsenescence, apoptosis, or malignancy. O'Sullivan and Karlseder (2010)Nat. Rev. Mol. Cell Biol. 11:171-181; Calado et al. (2012) Leukemia26:700-707; Artandi and DePinho (2010) Carcinogenesis 31:9-18.

The enzyme complex telomerase extends telomeres and comprises twoessential components: the telomerase reverse transcriptase (TERT), andan RNA component known as telomerase RNA component (TERC). Othercomponents of the telomerase complex include the proteins TCAB1,Dyskerin, Garl, Nhp2, Nop10, and RHAU. Brouilette et al. (2003)Arteriosclerosis, Thrombosis, and Vascular Biology 23:842-846. TERT is alimiting component of the telomerase complex, and thus treatments thatincrease TERT can increase telomerase activity. Telomerase activity istypically measured using the telomeric repeat amplification protocol(TRAP) assay, which quantifies the ability of a cell lysate or othersample to extend a synthetic telomere-like DNA sequence.

As would be expected due to the importance of telomere lengthmaintenance in preventing cellular senescence and apoptosis andresulting cellular dysfunction, genetic mutations of TERT and TERC arelinked to fatal inherited diseases of inadequate telomere maintenance,including forms of idiopathic pulmonary fibrosis, dyskeratosiscongenita, and aplastic anemia. The effects of premature cellularsenescence and apoptosis due to short telomeres in these diseases aredevastating in themselves, and may be compounded by increased risk ofcancer. Artandi and DePinho (2010) Carcinogenesis 31:9-18; Alter et al.(2009) Blood 113:6549-6557. In addition to abundant correlative datalinking short telomeres to cancer (Wentzensen et al. (2011) CancerEpidemiol. Biomarkers Prev. 20:1238-1250), aplastic anemia provides someof the first direct evidence that critically short telomeres andresulting chromosomal instability predispose cells to malignanttransformation in humans (Calado et al. (2012) Leukemia 26:700-707).There is evidence that short telomeres make the difference between fataland non-fatal muscular dystrophy (Sacco et al. (2010) Cell143:1059-1071), and that telomere extension averts endothelial cellsenescence (Matsushita et al. (2001) Circ. Res. 89:793-798), which isassociated with atherosclerosis, hypertension, and heart disease(Perez-Rivero et al. (2006) Circulation 114:309-317). In addition tobeing implicated in these and other diseases, short telomeres also limitcell amplification for cell therapies and bioengineering applications.Mohsin et al. (2012) Journal of the American College of Cardiologydoi:10.1016/j.jacc.2012.04.0474.

A natural product-derived telomerase activator, TA-65®, has been soldcommercially as a nutraceutical by T.A. Sciences, Inc. Harley et al.(2011) Rejuvenation Research 14:45-56. This compound purportedly turnson the endogenous hTERT gene, thus activating expression of nativetelomerase. It is not clear, however, how this treatment overcomes thenormal regulation of the native telomerase activity.

Human cells with little or no telomerase activity have been transfectedwith vectors encoding human TERT (hTERT). See, e.g., Bodnar et al.(1998) Science 279:349-352. The transfected cells were found to expresstelomerase, to display elongated telomeres, to divide vigorously, and todisplay reduced senescence compared to cells lacking telomerase, but thegenomic modification resulting from this treatment increases the riskand limits the utility of the approach.

A limited capacity to replicate is one of the defining characteristicsof most normal cells. An end-point of this limited replicative processis senescence, an arrested state in which the cell remains viable but nolonger divides. Senescent cells are often characterized by an alteredpattern of gene expression, altered morphology, and reduced or abrogatedability to perform their normal functions.

The shortening of telomeres plays a direct role in cellular senescencein animal tissues during aging. Furthermore, there is accumulatingevidence implicating short telomeres in a variety of diseases, includingthose described above. The prospect of preventing disease by telomereextension motivates a need for safe and effective treatments to extendtelomeres in animal cells in vivo and/or in vitro. Further, there is aneed to safely and rapidly extend telomeres in cells for use in celltherapy, cell and tissue engineering, and regenerative medicine.

At the same time, however, there is a danger in the constitutiveactivation of telomerase activity. Indeed for cell therapy applications,avoiding the risk of cell immortalization is of paramount importance. Tothis end, transient, rather than constitutive, telomerase activity maybe advantageous for safety, especially if the elevated telomeraseactivity is not only brief but extends telomeres rapidly enough that thetreatment does not need to be repeated continuously. Current methods ofextending telomeres include viral delivery of TERT under an induciblepromoter, delivery of TERT using vectors based on adenovirus andadeno-associated virus, and small molecule activators of telomerase.These methods risk either insertional mutagenesis, continual elevationof telomerase activity, or both.

Thus, there is strong motivation to develop a therapy that safelyextends telomeres to potentially prevent, delay, ameliorate, or treatthese and other conditions and diseases, to do the same for the gradualdecline in physical form and function and mental function thataccompanies chronological aging, and to enable cell therapies andregenerative medicine. Such a therapy would be of great use in therejuvenation of all animals, including humans, pets, livestock, zooanimals, and animals of endangered species.

SUMMARY OF THE INVENTION

The present invention addresses these and other problems by providing analternative that offers the benefits of a highly transient increase intelomerase activity, combined with rapid telomere extension so that thetreatment does not need to be continuous or even frequent, and with norisk of genomic insertional mutagenesis. Specifically, the inventionprovides compositions, methods, and kits for the extension of telomeresby the transient translation of exogenous telomerase activity in a cell.

In one aspect, the invention provides compounds for the extension oftelomeres comprising:

a synthetic ribonucleic acid comprising at least one modified nucleosideand coding for a telomerase reverse transcriptase;

wherein telomeres are extended within a cell treated with the compound.

In some embodiments, the telomerase reverse transcriptase is amammalian, avian, reptilian, or fish telomerase reverse transcriptase ora variant that retains telomerase catalytic activity, and in specificembodiments is a human telomerase reverse transcriptase.

In some embodiments, the ribonucleic acid comprises a 5′ cap, a 5′untranslated region, a 3′ untranslated region, and a poly-A tail. The 5′cap may be non-immunogenic and the 5′ cap may have been treated withphosphatase.

In preferred embodiments, the poly-A tail increases stability of theribonucleic acid.

In other preferred embodiments, the 5′ untranslated region or the 3′untranslated region comprise a sequence from a stable mRNA or an mRNAthat is efficiently translated, or they both comprise a sequence from astable mRNA or an mRNA that is efficiently translated.

In still other preferred embodiments, the 5′ cap, the 5′ untranslatedregion, or the 3′ untranslated region stabilizes the ribonucleic acid,increases the rate of translation of the ribonucleic acid, or modulatesthe immunogenicity of the ribonucleic acid.

In highly preferred embodiments, the at least one modified nucleosidemodulates immunogenicity of the ribonucleic acid.

In some embodiments, the ribonucleic acid is a purified syntheticribonucleic acid.

In preferred embodiments, the synthetic ribonucleic acid is purified toremove immunogenic components.

In certain specific embodiments, the ribonucleic acid codes for a human,cat, dog, mouse, horse, cow, sheep, pig, African elephant, chicken, rat,zebrafish, Japanese medaka, or chimpanzee telomerase reversetranscriptase, or a polypeptide with at least 95% sequence identity tothe telomerase reverse transcriptase.

In another aspect, the invention provides compositions comprising acompound of the invention and a telomerase RNA component, which, in someembodiments, is a mammalian, avian, reptilian, or fish telomerase RNAcomponent. In more specific embodiments, the telomerase RNA component isa human telomerase RNA component. In some embodiments, the compounds andcompositions of the invention further comprise a delivery vehicle.

In some embodiments, the delivery vehicle is an exosome, a lipidnanoparticle, a polymeric nanoparticle, a natural or artificiallipoprotein particle, a cationic lipid, a protein, a protein-nucleicacid complex, a liposome, a virosome, or a polymer. In specificembodiments, the delivery vehicle is a cationic lipid.

In preferred embodiments, the delivery vehicle is non-immunogenic. Inother preferred embodiments, the delivery vehicle is partly immunogenic.In particular, under some circumstances, it may be desirable for thevehicle to retain some immunogenicity.

According to another aspect of the invention, methods of extendingtelomeres are provided, comprising the step of administering any of thedisclosed compounds or compositions to an animal cell, wherein at leastone telomere is extended within the cell.

In some method embodiments, the cell has at least one shortened telomereprior to the administering step.

In some embodiments, the cell is from or in a subject suffering from orat risk of an age-related illness, an age-related condition, or anage-related decline in function or appearance.

In some embodiments, the cell is from or in a subject suffering from orat risk of cancer, heart disease, stroke, diabetes, diabetic ulcers,Alzheimer's disease, osteoporosis, a decline in physical ability orappearance, physical trauma or chronic physical stress, psychologicaltrauma or chronic psychological stress, reduced immune function,immunosenescence, or macular degeneration.

In some embodiments, the cell is a somatic cell of endodermal,mesodermal, or ectodermal lineage, or a germ line or embryonic cell.

In some embodiments, the cell is an induced pluripotent stem cell or acell used to produce an induced pluripotent stem cell.

In some embodiments, the cell is a transdifferentiated cell or a cellused to produce a transdifferentiated cell.

In some embodiments, the cell is an isolated cell, and the administeringstep lasts no longer than 48 hours. In other embodiments, the cell is anisolated cell, and the administering step lasts at least 2 hours.

In some embodiments, the cell is an isolated cell, and the administeringstep is performed no more than four times. In other embodiments, thecell is an isolated cell, and the administering step is performed atleast two times.

In some embodiments, the cell is an isolated cell, and the methodfurther comprises the step of measuring telomerase activity in the cell.In specific embodiments, the administering step increases telomeraseactivity in the cell, and in even more specific embodiments, thetelomerase activity is transiently increased by at least 5%. In otherspecific embodiments, the half-life of increased telomerase activity isno longer than 48 hours.

In some embodiments, the method further comprises the step of measuringtelomere length in the cell. In specific embodiments, average telomerelength is increased by at least 0.1 kb.

In some embodiments, the cell is an isolated cell, and the methodfurther comprises the step of measuring population doubling capacity inthe cell. In specific embodiments, the population doubling capacityincreases, in some cases by at least one population doubling.

In preferred embodiments, the cell is from or in a mammalian subject,and in even more preferred embodiments, the cell is from or in a humansubject.

In some embodiments, the cell is an isolated cell, and in otherembodiments, the cell is not an isolated cell. In some embodiments, theadministering step comprises electroporation. In some embodiments, theat least one telomere is transiently extended within the cell.

According to yet another aspect of the invention, kits for extendingtelomeres in an animal cell are provided, the kits comprising any of theabove compounds or compositions and instructions for using the compoundor composition to extend telomeres.

In some embodiments, the kits further comprise packaging materials. Insome specific embodiments, the packaging materials are air-tight. Insome specific embodiments, the packaging materials comprise a metal foilcontainer.

In some embodiments, the kits further comprise a desiccant, a culturemedium, or an RNase inhibitor.

In some embodiments, the composition is sterile. In some embodiments,the kit comprises a compound of the invention, instructions for usingthe composition to extend telomeres, and a telomerase RNA component, adelivery vehicle, or both a telomerase RNA component and a deliveryvehicle.

In yet another aspect, the invention provides methods of treatment,comprising administering a compound or composition of the invention toan animal subject in need of, or that may benefit from, telomereextension. In some embodiments, the animal subject suffers from or is atrisk of a disease or condition resulting from shortened telomeres.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Transfection of MRC-5 cells with a modified ribonucleic acid(modRNA) encoding TERT, but not catalytically-inactive TERT (CI TERT),transiently increases telomerase activity. (a) Schematic of the modRNAconstruct and approach used in these studies. The modRNA constructencodes human TERT and has 5′ and 3′ UTRs that confer stability, such asfrom β-globin mRNA. CI TERT has a single by mutation. (b) Transfectionof TERT or CI TERT modRNA results in increased exogenous TERT modRNAlevels in treated cells, but not untreated controls, above endogenousTERT RNA levels. (c) Levels of protein recognized by anti-TERT antibodyare significantly higher in MRC-5 cells 24 hours after transfection withmodRNA encoding TERT or CI TERT (P<0.03 or 0.01, respectively) than inuntreated or vehicle-only cells. (d) Treatment with modRNA encodingTERT, but not CI TERT, causes transient increase in telomerase activityin MRC-5 cells.

FIG. 2. Use of exosomes as a delivery vehicle for ribonucleic acidtherapeutics.

FIG. 3. Graphical illustration of the use of multiple rapid andtransient telomerase treatments in the extension of telomeres in a cell.

FIG. 4. Treatment with TERT modRNA rapidly extends telomeres in MRC-5cells. (a) Treatment and assay schedule used in these studies.Treatments lasted 5 hours. (b) Image of TRF Southern blot (inset) andits quantification in which the chemoluminescent signal was normalizedto account for the number of probes per unit of telomere length, and theaverage intensity of each pixel row in each gel lane was then plottedrelative to telomere length (n=3 biological replicates for eachtreatment). (c) Averages of telomere length distributions in FIG. 4b ,revealing that telomeres in cells treated with TERT modRNA aresignificantly (P<0.014) longer than in untreated or vehicle only-treatedcontrols. (d) Proportion of telomeres longer than an arbitrary threshold(4.2 kb) is greater in cells treated with TERT modRNA than in untreatedand vehicle only-treated controls. (e) Representative fluorescencemicrograph of metaphase spread of MRC-5 fibroblasts used in quantitativein situ hybridization (Q-FISH) to compare telomere lengths, showingtelomere probe (light, punctate) and DNA (smooth shading). (f)Quantification of Q-FISH measurements of telomere length in treated andcontrol cells (n=15 cells for each of two biological replicates for eachtreatment), showing rapid telomere extension in treated cells. (Notethat TRF measures the length of both telomeric and subtelomeric DNA,whereas Q-FISH measures only telomeric DNA, thus explaining thedifferences between the Q-FISH and TRF results.) (g) (i) Standard curverelating cumulative population doublings of MRC-5 cells followingreceipt from the supplier to telomere length as measured using Q-FISH toquantify average total telomere probe fluorescence per cell, which islinearly proportional to telomere length. (g) (ii) Quantification ofaverage telomere length per cell in MRC-5 cells treated three times withTERT modRNA at 48 hour intervals, as measured using Q-FISH.

FIG. 5. Brief treatment with TERT modRNA increases replicative capacityof, but does not immortalize, MRC-5 cells in a dose-dependent manner.(a) Growth curves of cells treated with TERT modRNA once, twice, orthree times, or three times followed by an additional treatment 8 weeksafter the first treatment. Controls comprise either no treatment, ortreatment with vehicle only or CI TERT four times. (n=3 for eachtreatment). (b) Growth curves of cells treated with TERT modRNA and TERCRNA in a 1:5 molar ratio (n=3). (c) Treatment once, twice, or threetimes with TERT modRNA confers additional replicative capacity to MRC-5cells beyond that of untreated cells, in a dose-dependent manner. Theincremental increase in proliferative capacity conferred by the secondand third treatments, delivered at 48 hours after the first treatment,is not as great as the increase in proliferative capacity conferred bythe first treatment; however, an additional fourth treatment severalweeks after the first three treatments confers as much additionalproliferative capacity as did the first treatment, indicating that thetiming of the treatments is important for optimizing the amount ofproliferative capacity conferred by the treatment. Treatment three timeswith TERT modRNA and TERC modRNA together in a 1:5 molar ratio confersgreater proliferative capacity than treatment three times with TERTmodRNA alone. Treatment with vehicle only or CI TERT modRNA confers noadditional replicative capacity. (n=3).

FIG. 6. High efficiency of transfection of MRC-5 cells treated withmodRNA encoding nuclear GFP (nGFP). (a) Percentage of cells withfluorescence more than two SD above the mean of untreated cells(n=10000). (b) Mean fluorescence (n=10000). (c) Fluorescence micrographof nGFP in transfected MRC-5 cells, counterstained with DAPI.

FIG. 7. Dose-response of telomerase activity.

FIG. 8. TERT modRNA treatment delays cell swelling in MRC-5 cells. (a)MRC-5 cells in early passages (left) are several times smaller area thancells in later passages (right), as shown in typical micrographs of PD 2and PD 53 untreated cells as seen on a hemocytometer. (b) Early (PD 2)and mid (PD 35) passage MRC-5 cells exhibit little swelling, defined ashaving a diameter greater than 25 microns as measured visually on ahemocytometer (where one small square is 50 microns across), whereas asignificantly greater fraction of late passage (PD 53) cells areswollen. In contrast, PD 53 cells that had been treated with TERTmodRNA, but not those treated with CI TERT modRNA, at PD 40 exhibitlittle swelling.

FIG. 9. Growth curve for human microvascular dermal endothelial cells(HMDECs).

FIG. 10. Effect of TERT modRNA treatment on cell number in HMDECs. CO:control treatment; hTERT-CI: catalytically-inactive hTERT; hTERT-WT:wild-type hTERT.

FIG. 11. Effect of TERT modRNA treatment on growth of HMDECs. UT:untreated; CO: control treatment; CI: catalytically-inactive hTERT; WT:wild-type hTERT.

FIG. 12. Effect of TERT modRNA treatment on senescence in HMDECs. NT:untreated; hTERT-CI: catalytically-inactive hTERT; hTERT-WT: wild-typehTERT.

FIG. 13. Increases in TERT protein and telomerase activity followingmodified TERT mRNA delivery. (A) Schematic of modified mRNA comprisingthe coding sequence of the full length functional form of TERT or acatalytically-inactive (CI) form of TERT, flanked by untranslatedregions (UTRs) of HBB and a 151 nt poly-A tail, synthesized usingmodified nucleotides pseudouridine and 5-methylcytidine. (B)Transfection efficiency of myoblasts (n=2,000) treated with 0.8 μg/mlmodified mRNA encoding GFP measured by flow cytometry 24 hpost-transfection exceeded 95% (additional plots in FIG. 16A). (C) TotalTERT protein levels were measured by quantitative Western blot (panel C,left; FIG. 17). Quantification of TERT protein levels 24 hours aftertransfection with 1 μg/ml of either TERT or CI TERT mRNA (n=3).Quantification of TERT protein in response to various doses of mRNA wasmeasured at the single cell level by flow cytometry (n=10,000) (panel C,right). *P<0.05, **P<0.01 compared to untreated cells. Error barsrepresent s.e.m. (D) Detection of telomerase activity in fibroblasts andmyoblasts transfected with 1 μg/ml modified TERT mRNA, as measured usingthe telomere repeat amplification protocol (TRAP). Arrow indicatesinternal controls for PCR efficiency.

FIG. 14. Increases in telomere length and proliferative capacityfollowing modified TERT mRNA (modRNA) delivery. (A) Mean telomerelengths in untreated fibroblasts decreased over time in culture asmeasured by MMqPCR and by SpectraCell (correlation coefficient 0.97,P<0.001). Experiment was repeated twice with four technical replicateseach. (B) Mean telomere lengths in fibroblasts transfected with 1 μg/mlTERT modRNA, CI TERT mRNA, or vehicle only, once, twice, or three timesin succession at 48 h intervals. Experiment was repeated twice with fourtechnical replicates each. **P<0.01, ***P<0.001 compared to vehicleonly-treated cells. (C) Mean telomere lengths in myoblasts treated as in(B). Experiment was repeated twice with four technical replicates each.***P<0.001 compared to vehicle only-treated cells. (D) Growth curves offibroblasts treated as in (B), with green arrows indicating treatmentstimes. Growth curves were repeated twice with each population culturedin triplicate. Replicative capacity increased in a dose-dependent manner(right panel). *P<0.05, **P<0.01 compared to vehicle only-treated cells.(E) Proliferation capacity of myoblasts, treated as in (B) (greenarrows). Growth curves were repeated twice, with each populationcultured in triplicate. All data are presented as means±s.e.m.

FIG. 15. Transient reduction of senescence-associated markers followingmodified TERT mRNA delivery. (A) Quantification of β-gal-expressingfibroblasts after modified TERT mRNA transfection three times insuccession at 48 h intervals (green arrows). The control cells,comprising untreated, vehicle only, and CI TERT mRNA-treatedpopulations, stopped expanding at PD 53, and the TERT modRNA-treatedpopulation stopped expanding at PD 80. Each experiment was conductedtwice with >50 cells per sample scored manually. Representative imagesshow β-gal-stained TERT modRNA-treated fibroblasts at PD 53 (top) and PD80 (bottom). Scale bar length is 200 microns. (B) Quantification ofβ-gal expression in myoblasts treated as in (A). Controls are as in (A).The control and TERT modRNA-treated populations stopped expanding at PD8 and PD 11, respectively. Each experiment was conducted twice, with >50cells per sample scored manually. Representative images show myoblastsat PD 2 (top) and TERT modRNA-treated myoblasts at PD 11 (bottom). (C)Quantification of enlarged cells associated with replicative senescencein fibroblasts transfected three times with modified TERT mRNA.Population plateaus are as in (A). Controls are vehicle only and CI TERTmRNA-treated. Each experiment was conducted twice, with >50 cells persample scored manually. Representative images show untreated fibroblastsat PD 2 (top) and PD 53 (bottom). All data are presented as means±s.e.m.Scale bar length is 200 microns.

FIG. 16. High efficiency transfection of modified mRNA into humanmyoblasts. (A,B) Quantification of GFP fluorescent myoblasts (n=2000)transfected with 0.1-0.8 μg/ml of modified mRNA encoding GFP as measuredby flow cytometry 24 h after start of treatment. (C) Mean fluorescenceof modified GFP mRNA-transfected myoblasts in response to increasingdoses. (D) Quantification of exogenous TERT modRNA in fibroblasts 24 hafter transfection with 1 μg/ml of TERT or CI TERT modRNA, as measuredusing RT-qPCR. Ratio of TERT to CI TERT was calculated using the Pfafflmethod with RPL37A and GAPDH as reference genes (n=3). (E)Quantification of endogenous TERT modRNA in fibroblasts 24 h aftertransfection with 1 μg/ml of TERT or CI TERT modRNA, as measured usingqPCR, calculated as in (D). All data are presented as means±s.e.m.

FIG. 17. Expression of TERT after modified mRNA delivery. TERT proteinexpression in fibroblasts harvested 24 h after start of treatment with 1μg/ml TERT modRNA was measured by multiplexed infrared Western blot. Theserial dilution of total protein was used to generate a standard curveto compare relative amounts of TERT protein to controls. The specificityof the TERT antibody used here has been extensively tested (Wu, 2006).

FIG. 18. TRAP gel showing increased telomerase activity in mononuclearcells electroporated with TERT modRNA at a setting of 200 V, 25 uF, 1000Ohm, in a 1 mm cuvette with 10 ul of cell suspension and TERT modRNA.The heat treated sample lane contains a primer dimer artifact of themethod as indicated by the strong band at the fourth rung of the ladder.

FIG. 19. Microscope images of mononuclear cells fluorescing afterelectroporation with modRNA encoding GFP.

FIG. 20. Electroporation of GFP modRNA into human leukocytes(mononuclear cells) results in high transfection efficiency.

FIG. 21. Image of gel from TRAP assay showing increasing telomeraseactivity in fibroblasts electroporated with increasing doses of TERTmodRNA.

FIG. 22. Fluorescent micrographs showing that CD8+ T-cells activatedwith CD3/CD28 and electroporated with GFP modRNA express GFP activity.Dark dots are CD3/CD28-coated beads.

FIG. 23. Electroporation of human keratinocytes with TERT modRNA resultsin expression of telomerase activity.

FIG. 24. Expression of a luciferase modRNA after in vivo delivery to arodent.

DETAILED DESCRIPTION OF THE INVENTION

Telomeres are DNA sequences at the ends of chromosomes that protect theends of the chromosomes but that shorten over time. Critically shorttelomeres may cause cells to stop functioning correctly or to die.Critically short telomeres may also lead to chromosome fusions that mayin turn lead to cancer. Even in the absence of a specific diagnoseddisease, short telomeres are implicated in the gradual decline infunction of the mind and body and in the appearance of aging.

At the same time, however, telomere shortening can play a protectiverole against cancer, for example in the situation where a cell acquiresa mutation that causes it to profilerate faster than normal cells. Inthat situation, telomere shortening prevents the cell from proliferatingindefinitely and causing cancer. It is therefore not necessarilybeneficial to extend telomeres continually.

In mammals, telomeres comprise tandem repeats of the sequence TTAGGG,and in other animals such as birds, reptiles, and fish, the repeatedsequence varies. In all of these types of animals, the telomeres aredouble stranded for many kilobases (kb). Average telomere lengths differbetween species as shown in Table 1.

TABLE 1 Average telomere length in adult fibroblasts of differentspecies Average telomere Species length (kb) Cow 18 Sheep 18 Pig 15Horse 14 Dog 15 Panda 25 Tiger 50 House mouse 40 Sonoran deer mouse 9Norway rat 40 Naked mole rat 16 European white rabbit 50 Black-tailedjack rabbit 25 Spider monkey 7 Squirrel monkey 9 Rhesus monkey 16Orangutang 10 Bonobo 10 Human 9 Indian elephant 15 African elephant 14Cat 11

In humans, telomeres start out before birth with lengths of 15-20 kb,and at birth with lengths of 12-15 kb. Telomeres shorten rapidly duringchildhood, and then by about 0-100 bp per year on average in adulthood,a rate which varies depending on the cell type, exposure topsychological or oxidative stress, and other factors.

Telomeres are part of the telomere complex, which protects the ends ofchromosomes. The telomere complex also comprises a set of proteinscollectively called Shelterin. Telomere complex proteins include POT1,TPP1, ATM, DAT, TRF1, TRF2, Rap1, Rif1, TIN2, NBS, MRE17, and RAD50 andtheir homologs in different mammalian species. Podlevsky and Chen (2012)Mutat. Res. 730:3-11. In many species the telomere terminates in asingle-stranded 3′ overhang which inserts itself into the doublestranded region, in association with telomere complex proteins, forminga loop within the telomere complex.

Telomeres shorten over time, due to oxidative damage and sisterchromatid exchange, and also due to the end replication problem, inwhich the ends of chromosomes are not completely duplicated duringmitosis. When telomeres become critically short, the telomere complex isno longer able to protect the chromosome ends, and the chromosome endsbecome “uncapped”. Uncapping of the chromosome ends may result inchromosome-chromosome fusions, which may in turn result in cancer.O'Sullivan and Karlseder (2010) Nat. Rev. Mol. Cell Biol. 11:171-181;Calado et al. (2012) Leukemia 26:700-707; Artandi and DePinho (2010)Carcinogenesis 31:9-18. Uncapping can also result in the chromosome endsbeing recognized as damaged DNA, activating DNA damage responses andtriggering cell apoptosis or senescence. Senescence is an arrested statein which the cell remains viable but no longer divides, and senescentcells typically cease to perform their normal, pre-senescence, usefulfunctions adequately or at all. Thus telomere shortening leads to tissuedysfunction, loss of physical ability and youthful appearance, loss ofmental ability, and disease in part due to the accumulation of senescentcells, and in part due to the loss of cells by apoptosis. Indeed, agedpeople with short telomeres are approximately 200-750% more likely todevelop myocardial infarction (200%) (von Zglinicki et al. (2000)Laboratory Investigation; a Journal of Technical Methods and Pathology80:1739-1747), vascular dementia (200%) (Testa et al. (2011) DiabeticMedicine 28:1388-1394, diabetes with complications (400%) (Blackburn etal. (2010) Cancer Prevention Research 3:394-402, cancer (Stern and Bryan(2008) Cytogenetic and Genome Research 122:243-254), stroke, Alzheimer'sdisease, infection (750%), idiopathic pulmonary fibrosis, and otherdisease. People with short telomeres in one tissue are likely to alsohave short telomeres in most of their other tissues, and thus shorttelomeres correlate with increased risk for many diseases in oneindividual. Takubo et al. (2010) Geriatr Gerontol Int. 10 Suppl1:S197-206; doi: 10.1111/j.1447-0594.2010.00605.x. Short telomeres alsolimit cell replicative capacity which in turn limits cell therapies andregenerative therapies. Conversely, increasing telomere length in micewith short telomeres using virus-based genetic engineering methodsrejuvenates the mice by several parameters, including skin thickness andelasticity, liver function, and sense of smell. Jaskelioff (2011) Nature469:102-107.

Since telomerase extends telomeres, a useful approach to extendingtelomeres is to increase the level of telomerase activity in cells. Manyagents and conditions have been reported to increase telomerase activityin cells, including the agents and conditions listed in Table 2.

TABLE 2 Examples of agents and conditions that increase telomeraseactivity Type Examples Growth factors EGF, IGF-1, FGF-2, VEGF (Liu etal. (2010) Ageing Research Reviews 9: 245-256) Genetic treatments Viraldelivery of DNA encoding TERT (Matsushita (2001) Circ. Res. 89: 793-798;Hooijberg (2000) J. Immunol. 165: 4239-4245); electroporation of plasmidencoding TERT (Bodnar et al. (1998) Science 279: 349-352); transfectionof mRNA encoding TERT (Seebe-Larssen et al. (2002) J. Immunol. Methods259:191-203) Hormones Estrogen (Imanishi et al. (2005) Journal ofHypertension 23: 1699-1706), erythropoietin (Akiyama et al. (2011)Leukemia Research 35: 416-418) Physical treatments UV radiation (Ueda etal. (1997) Cancer Research 57: 370- 374), hypoxia (Gladych et al. (2011)Biochemistry and Cell Biology 89: 359-376) Cytokines IL-2, IL-4, IL-6,IL-7, IL-13, and IL-15 (Liu et al. (2010) Ageing Research Reviews 9:245-256) Small molecules from plants Resveratrol (Pearce et al. (2008)Oncogene 27: 2365-2374), compounds extracted from Astragalusmembranaceus including cycloastragenol (TAT2), TA-65, or TAT153 (Zverevaet al. (2010) Biochemistry.  

  75: 1563- 1583; Harley et al. (2011) Rejuvenation Research 14: 45-56)Other Inhibitors of Menin, SIP1 (Lin and Elledge (2003) Cell 113:881-889), pRB, p38 (Di Mitri et al. (2011) Journal of Immunology 187:2093-2100, p53, p73 (Beitzinger et al. (2006) Oncogene 25:813-826,MKRN1, CHIP, Hsp70 (Lee et al. (2010) The Journal of BiologicalChemistry 285: 42033- 42045), androgens (Nicholls et al. (2011) Protein& Cell 2: 726-738), and TGF-beta (Prade-Houdellier et al. (2007)Leukemia 21: 2304-2310)

The treatment examples of Table 2 are not without undesired effects,however. For example, treatment with growth factors, hormones, orcytokines may cause side effects, may activate multiple signalingpathways, may cause unwanted cell replication, may trigger an immuneresponse, and are generally non-specific. Genetic treatments usingplasmids or viruses carry a risk of genomic modification by insertionalmutagenesis and a risk of cancer. Transfection with unmodified RNAcauses a strong immune response and has not been shown to extendtelomeres. Physical treatments can damage genomic DNA. Treatment withsmall molecules from plants have been found to only extend telomeres insome subjects and cells, only extend telomeres very slowly, and requirechronic delivery, therefore risking cancer.

The expression in cells of nucleic acid sequences encoding hTERT andTERC, and the use of these components themselves, have been proposed tobe useful in the diagnosis, prognosis, and treatment of human diseases(see, e.g., U.S. Pat. Nos. 5,583,016 and 6,166,178), but telomereextension in a manner that is both rapid and transient, and thuspotentially safe for the reasons described above, has not beendemonstrated. Sæbe-Larssen et al. (2002) J. Immunol. Methods 259:191-203reported the transfection of dendritic cells with mRNA encoding hTERT,and that such cells acquired telomerase activity, but the transfectionused standard mRNA and resulted in a strong hTERT cytotoxic T lymphocyte(CTL) response rather than an extension of telomeres.

Furthermore, all existing small-molecule treatments are largelyineffective and slow (Harley et al. (2011) Rejuvenation Research14:45-56), primarily because they act through the catalytic component oftelomerase, TERT, which is heavily regulated post-translationally,limiting existing treatments' effects to a small subset of cells, andexcluding cells in interphase or G0 such as many stem and progenitorcells. Cifuentes-Rojas and Shippen (2012) Mutat. Res. 730:20-27;doi:10.1016/j.mrfmmm.2011.10.003; Cong et al. (2002) Microbiology andMolecular Biology Reviews 66:407-425. This regulation is mediated inpart by interactions between components of the telomerase complex, thetelomere complex, and other molecules. For example, TERT isphosphorylated or dephosphorylated at multiple sites by multiple kinasesand phosphatases, and at some sites, phosphorylation results inincreased telomerase activity (for example phosphorylation by Akt),while at others sites phosphorylation reduces telomerase activity (forexample, phosphorylation by Srcl or cAbl). Also, TERT is ubiquitinatedor deubiquitinated at specific sites. TERT also interacts with otherproteins at specific sites on TERT, and these interactions caninactivate TERT (for example interactions with Pinxl or cAbl), ortransport TERT away from the chromosomes (for example, interactions withCRM1 and Pinxl), preventing or slowing telomere extension. Further, someproteins bind to telomeres or the telomere complex, blocking TERT (forexample POT1), preventing telomere extension. Further, some proteins aidtelomere extension indirectly, for example helicases and UPF1. Due toregulatory mechanisms, telomerase activity peaks during S phase of thecell cycle, and thus rapidly-dividing cells may tend to benefit morefrom treatments that increase telomerase activity. However, it is oftendesirable to keep cells in a slow-dividing or non-dividing state; forexample, stem or progenitor cells are often slow-dividing, and thus mayspend the majority of their time in interphase or G₀. Thus, existingtreatments are slow and ineffective in most cell types generally, and inall cell types during interphase and G₀. Treatments that are slow areless safe, because they require treatment for a longer time. Sincetelomere-shortening provides a protective safety mechanism againstrun-away cell proliferation, such as in cancer, a treatment that extendstelomeres rapidly is generally safer, because it may be delivered forshort periods of time and infrequently, thus allowing the normaltelomere-shortening safety mechanism to remain in effect for much of thetime. Therefore a method capable of transiently overcoming telomeraseregulation to rapidly extend telomeres during a brief treatment isneeded.

TERT regulates hundreds of genes including those listed in Table 3.

TABLE 3 Examples of genes and pathways regulated by TERT Type ExamplesUpregulated Epigenetic state modulators DNA 5-methylcytosine transferaseI (Young et al. (2003) The Journal of Biological Chemistry 278:19904-19908) Proto-oncogenes Hepatocyte growth factor receptor (MET),AKT-2, CRK (Perrault et al. (2005) Biochemical and Biophysical ResearchCommunications 335: 925-936) Differentiation, cell fate Sox-13 (Perraultet al. (2005) Biochemical and Biophysical Research Communications 335:925-936), Wnt (Park et al. (2009) Nature 460: 66-72) GlycolysisPhosphofructokinase (Bagheri et al. (2006) Proc. Nat'l Acad. Sci. U.S.A.103: 11306-11311), aldolase C (Bagheri et al. (2006) Proc. Nat'l Acad.Sci. U.S.A. 103: 11306-11311) Proliferation enhancers Activatingtranscription factor-3, Xbox protein-1, FGF, EGFR (Smith et al. (2003)Nature Cell Biology 5: 474-479), Insulin- like growth factor 2 (Perraultet al. (2005) Biochemical and Biophysical Research Communications 335:925-936), Wnt (Park et al. (2009) Nature 460: 66-72), tp53bp1 (Perraultet al. (2005) Biochemical and Biophysical Research Communications 335:925-936), epiregulin (Lindvall et al. (2003) Cancer Research 63:1743-1747) Metastasis-related genes Mac-2 binding protein (Park et al.(2007) International Journal of Cancer 120: 813-820) DownregulatedProliferation inhibitors Interleukin 1 receptor antagonist, parathyroidhormone-related peptide, integrin-associated protein, TNF-relatedapoptosis- inducing ligand (Smith et al. (2003) Nature Cell Biology 5:474-479), IGF binding protein-5 (Perrault et al. (2005) Biochemical andBiophysical Research Communications 335: 925-936), Melanoma inhibitoryactivity (Perrault et al. (2005) Biochemical and Biophysical ResearchCommunications 335: 925-936), p21, p53 Differentiation, cell fateTransforming growth factor B2 (Perrault et al. (2005) Biochemical andBiophysical Research Communications 335: 925-936)

In many cases, modulating the genes or pathways of Table 3 isundesirable because doing so can cause unwanted changes in cells. Forexample, TERT activates epigenetic regulators, which can change cellphenotype or interfere with efforts to reprogram or transdifferentiatecells for therapeutic purposes.

TERT activates growth enhancers, but often proliferation is not desired,for example often stem cells with the most regenerative potential arethose which divide slowly. TERT modulates regulators of cell fate anddifferentiation, which can impair efforts to differentiate cells intospecific cell types. TERT also activates proto-oncogenes, which couldlead to cancer. Thus, it is desirable to minimize the amount of timeduring which TERT levels are artificially elevated, including anytreatment that extends telomeres using TERT. A treatment that extendstelomeres by only transiently increasing telomerase activity levels istherefore needed.

In some cell types TERT has been shown to affect expression of othergenes (Young et al. (2003) J. Biol. Chem. 278:19904-19908; Perrault etal. (2005) Biochem. Biophys. Res. Commun. 335:925-936), and this may notbe desirable in some cases. Thus, a treatment that minimizes the amountof time during which TERT levels are increased is needed.

Compounds

The present invention addresses these problems by providing in oneaspect novel compounds for the transient expression of exogenoustelomerase in a cell. The compounds comprise a synthetic ribonucleicacid comprising at least one modified nucleoside and coding for atelomerase reverse transcriptase (TERT), wherein telomeres are extendedwithin a cell treated with the compound.

Synthetic Ribonucleic Acids

The ribonucleic acids used in the transient expression of TERT accordingto various aspects of the instant invention comprise a ribonucleic acidcoding for a TERT protein. The ribonucleic acids typically furthercomprise sequences that affect the expression and/or stability of theribonucleic acid in the cell. For example, as shown in FIG. 1a , theribonucleic acids may contain a 5′ cap and untranslated region (UTR) tothe 5′ and/or 3′ side of the coding sequence. The ribonucleic acids mayfurther contain a 3′ tail, such as a poly-A tail. The poly-A tail may,for example, increase the stability of the ribonucleic acid. In someembodiments, the poly-A tail is at least 75 nucleotides, 100nucleotides, 125 nucleotides, 150 nucleotides, or even longer.

In some embodiments, the 5′ cap of the ribonucleic acid is anon-immunogenic cap. In some embodiments, the 5′ cap may increase thetranslation of the ribonucleic acid. In some embodiments, the 5′ cap maybe treated with phosphatase to modulate the innate immunogenicity of theribonucleic acid. In some embodiments, the 5′ cap is an anti-reverse capanalog (“ARCA”), such as a 3′-O-Me-m7G(5′)ppp(5′)G RNA cap structureanalog.

As is well-known in the art, the above features, or others, may increasetranslation of the TERT protein encoded by the ribonucleic acid, mayimprove the stability of the ribonucleic acid itself, or may do both. Insome embodiments, the 5′ UTR and/or the 3′ UTR are from a gene that hasa very stable mRNA and/or an mRNA that is rapidly translated, forexample, α-globin or β-globin, c-fos, or tobacco etch virus. In someembodiments, the 5′ UTR and 3′ UTR are from different genes, or are fromdifferent species than the species into which the compositions are beingdelivered. The UTRs may also be assemblies of parts of UTRs from themRNAs of different genes, where the parts are selected to achieve acertain combination of stability and efficiency of translation.

The ribonucleic acids of the invention are preferablynucleoside-modified RNAs (“modRNA”). Most mature RNA molecules ineukaryotic cells contain nucleosides that are modified versions of thecanonical unmodified RNA nucleosides, adenine, cytidine, guanosine, anduridine. Those modifications may prevent the RNA from being recognizedas a foreign RNA. Karikó et al. (2005) Immunity 23:165-175. SyntheticRNA molecules made using certain nucleosides are much less immunogenicthan unmodified RNA. The immunogenicity can be reduced even further bypurifying the synthetic modRNA, for example by using high performanceliquid chromatography (HPLC). The modified nucleosides may be, forexample, chosen from the nucleosides shown in Table 4. The nucleosidesare, in some embodiments, pseudouridine, 2-thiouridine, or5-methylcytidine. Under some circumstances, it may be desirable for themodified RNA to retain some immunogenicity.

TABLE 4 Modified nucleosides found in eukaryotic RNA symbol common namem¹A 1-methyladenosine m⁶A N⁶-methyladenosine Am 2′-O-methyladenosine i⁶AN⁶-isopentenyladenosine io⁶A N⁶-(cis-hydroxyisopentenyl)adenosinems²io⁶A 2-methylthio-N⁶-(cis-hydroxyisopentenyl) adenosine g⁶AN⁶-glycinylcarbamoyladenosine t⁶A N⁶-threonylcarbamoyladenosine ms²t⁶A2-methylthio-N⁶-threonyl carbamoyladenosine Ar(p) 2′-O-ribosyladenosine(phosphate) m⁶ ₂A N⁶,N⁶-dimethyladenosine m⁶Am N⁶,2′-O-dimethyladenosinem⁶ ₂Am N⁶,N⁶,2′-O-trimethyladenosine m¹Am 1,2′-O-dimethyladenosine m³C3-methylcytidine m⁵C 5-methylcytidine Cm 2′-O-methylcytidine ac⁴CN⁴-acetylcytidine f⁵C 5-formylcytidine m⁴C N⁴-methylcytidine hm⁵C5-hydroxymethylcytidine f⁵Cm 5-formyl-2′-O-methylcytidine m¹G1-methylguanosine m²G N²-methylguanosine m⁷G 7-methylguanosine Gm2′-O-methylguanosine m² ₂G N²,N²-dimethylguanosine Gr(p)2′-O-ribosylguanosine (phosphate) yW wybutosine o₂yW peroxywybutosineOHyW hydroxywybutosine OHyW* undermodified hydroxywybutosine imG wyosinem^(2,7)G N²,7-dimethylguanosine m^(2,2,7)G N²,N²,7-trimethylguanosine Iinosine m¹I 1-methylinosine Im 2′-O-methylinosine Q queuosine galQgalactosyl-queuosine manQ mannosyl-queuosine Ψ pseudouridine Ddihydrouridine m⁵U 5-methyluridine Um 2′-O-methyluridine m⁵Um5,2′-O-dimethyluridine m¹Ψ 1-methylpseudouridine Ψm2′-O-methylpseudouridine s²U 2-thiouridine ho⁵U 5-hydroxyuridine chm⁵U5-(carboxyhydroxymethyl)uridine mchm⁵U 5-(carboxyhydroxymethyl)uridinemethyl ester mcm⁵U 5-methoxycarbonylmethyluridine mcm⁵Um5-methoxycarbonylmethyl-2′-O-methyluridine mcm⁵s²U5-methoxycarbonylmethyl-2-thiouridine ncm⁵U 5-carbamoylmethyluridinencm⁵Um 5-carbamoylmethyl-2'-O-methyluridine cmnm⁵U5-carboxymethylaminomethyluridine m³U 3-methyluridine m¹acp³Ψ1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine cm⁵U5-carboxymethyluridine m³Um 3,2′-O-dimethyluridine m⁵D5-methyldihydrouridine τm⁵U 5-taurinomethyluridine τm⁵s²U5-taurinomethyl-2-thiouridine

Without intending to be bound by theory, the presence of the modifiednucleosides enables modRNA to avoid activation of an immune responsemediated by various receptors, including the Toll-like receptors andRIG-1. Non-immunogenic modRNA has been used as a therapeutic agent inmice via topical delivery. Kormann et al. (2011) Nature Biotechnology29:154-157. The discovery of nucleotide-modified mRNA facilitates thedelivery of RNA-encoded therapeutic proteins, or mutants thereof, tocells, and the expression of those proteins in cells.

Accordingly, in some embodiments, the ribonucleic acids of the instantcompositions comprise a pseudouridine, a 2-thiouridine, a5-methylcytidine, or a nucleoside from Table 4. In some embodiments, theribonucleic acids comprise more than one of the above nucleosides orcombination of the above nucleosides. In highly preferred embodiments,the ribonucleic acids comprise pseudouridine and 5-methylcytidine.

In some embodiments, an immune response to the modRNA may be desired,and the RNA may be modified to induce an optimal level of innateimmunity. In other embodiments, an immune response to the modRNA may notbe desired, and the RNA may be modified in order to minimize such areaction. The RNA can be modified for either situation.

The ribonucleic acids of the instant invention are preferably syntheticribonucleic acids. The term “synthetic”, as used herein, means that theribonucleic acids are in some embodiments prepared using the tools ofmolecular biology under the direction of a human, for example asdescribed below. The synthetic ribonucleic acids may, for example, beprepared by in vitro synthesis using cellular extracts or purifiedenzymes and nucleic acid templates. The synthetic ribonucleic acids mayin some embodiments be prepared by chemical synthesis, either partiallyor completely. Alternatively, or in addition, the synthetic ribonucleicacids may in some embodiments be prepared by engineered expression in acell, followed by disruption of the cell and at least partialpurification of the ribonucleic acid. A synthetic ribonucleic acid isnot, however, a naturally-occurring ribonucleic acid, as it is expressedin an unmodified cell without extraction or purification.

The ribonucleic acids of the instant invention may be prepared using avariety of standard techniques, as would be understood by one ofordinary skill in the art. In some embodiments, the ribonucleic acidsmay be prepared by in vitro synthesis, as described, for example, inU.S. Patent Application Publication Nos. 2009/0286852 and 2011/0143397.In some embodiments, the ribonucleic acids may be prepared by chemicalsynthesis. In some embodiments, the ribonucleic acids may be prepared bya combination of in vitro synthesis and chemical synthesis. As describedabove, the term “synthetic” should be understood to include ribonucleicacids that are prepared either by chemical synthesis, by in vitrosynthesis, by expression in vivo and at least partial purification, orby a combination of such, or other, chemical or molecular biologicalmethods.

The ribonucleic acids of the instant invention may, in some embodiments,be purified. As noted above, purification may reduce immunogenicity ofthe ribonucleic acids and may be advantageous in some circumstances. Seealso U.S. Patent Application Publication No. 2011/0143397. In preferredembodiments, the ribonucleic acids are purified by HPLC or by affinitycapture and elution.

The protein structure of TERT includes at least three distinct domains:a long extension at the amino-terminus (the N-terminal extension, NTE)that contains conserved domains and an unstructured linker region; acatalytic reverse-transcriptase domain in the middle of the primarysequence that includes seven conserved RT motifs; and a short extensionat the carboxyl-terminus (the C-terminal extension, CTE). Autexier andLue (2006) Annu Rev Biochem. 75:493-517. In some embodiments, theribonucleic acid of the instant invention codes for a full-length TERT.In some embodiments, the ribonucleic acid codes for a catalytic reversetranscriptase domain of TERT. In some embodiments, the ribonucleic acidcodes for a polypeptide having TERT activity.

The TERT encoded by the ribonucleic acids of the instant disclosure ispreferably a mammalian, avian, reptilian, or fish TERT. More preferably,the TERT is a mammalian TERT, such as human TERT. Meyerson et al. (1997)Cell 90:785-795; Nakamura et al. (1997) Science 277:955-959; Wick et al.(1999) Gene 232:97-106. The amino acid sequence of two human TERTisoforms are available as NCBI Reference Sequences: NP_937983.2 andNP_001180305.1. Other non-limiting exemplary amino acid sequencesusefully encoded by the ribonucleic acids of the instant compositionsinclude TERT from cat (NCBI Reference Sequence: XP_003981636.1), dog(NCBI Reference Sequence: NP_001026800.1), mouse (NCBI ReferenceSequence: NP_033380.1), cow (NCBI Reference Sequence: NP_001039707.1),sheep NCBI Reference Sequence: XP_004017220.1), pig (NCBI ReferenceSequence: NP_001231229.1), African elephant (NCBI Reference Sequence:XP_003408191.1), chicken (NCBI Reference Sequence: NP_001026178.1), rat(NCBI Reference Sequence: NP_445875.1), zebrafish (NCBI ReferenceSequence: NP_001077335.1); Japanese medaka (NCBI Reference Sequence:NP_001098286.1); and chimpanzee (NCBI Reference Sequences:XP_003950543.1 and XP_003950544.1).

It should be understood that the ribonucleic acids of the instantinvention may code for variants of any of the above-listed amino acidsequences, particularly variants that retain telomerase catalyticactivity, including truncated variants. In some embodiments, theribonucleic acids of the instant compositions code for one of theabove-listed amino acid sequences or a sequence with at least 95%sequence identity to that sequence. In some embodiments, the nucleicacids of the instant compositions code for one of the above-listed aminoacid sequences or a sequence with at least 98%, 99%, 99.9%, or evenhigher sequence identity to that sequence.

It should also be understood that the instant ribonucleic acids maycorrespond to the native gene sequences coding for the above-listed TERTproteins or may correspond to variants that are made possible due to theredundancy of the genetic code, as would be understood by one ofordinary skill in the art. In some embodiments, the codon selection maybe optimized to optimize protein expression using algorithms and methodsknown by those of ordinary skill in the art. Fath et al. (2011) PLoS ONE6:3.

Compositions

In another aspect, the present invention provides compositions for theextension of telomeres in a cell, the compositions comprising a compoundof the invention, as described above, and a further component. In someembodiments, the compositions further comprise a telomerase RNAcomponent (TERC). (See also Table 6 below.) In some embodiments, thecompositions further comprise a delivery vehicle.

Delivery Vehicles

As just noted, the compositions of the instant disclosure may furthercomprise a delivery vehicle for the ribonucleic acid. The deliveryvehicle may, in some cases, facilitate targeting and uptake of theribonucleic acid of the composition to the target cell. In particular,the compositions of the instant disclosure may comprise any genedelivery vehicle known in the field, for example nanoparticles,liposomes, gene gun ballistic particles, viruses, cationic lipids,commercial products, such as Lipofectamine® RNAiMax, or other vehicles.In some embodiments, the delivery vehicle is an exosome, a lipidnanoparticle, a polymeric nanoparticle, a natural or artificiallipoprotein particle, a cationic lipid, a protein, a protein-nucleicacid complex, a liposome, a virosome, or a polymer. In some preferredembodiments, the delivery vehicle is a cationic lipid formulation. Viraldelivery is typically not preferred, however, as it can lead toinsertional mutagenesis.

In some preferred embodiments, the delivery vehicle is an exosome, alipid nanoparticle, or a polymeric nanoparticle. In highly preferredembodiments, the delivery vehicle is an exosome. Exosomes arenaturally-occurring lipid bilayer vesicles 40-100 nm in diameter.Exosomes contain a set of specific proteins, including the membraneprotein Lamp-1 and Lamp-2, which are particularly abundant. Lakhal andWood (2011) BioEssays: News and Reviews in Molecular, Cellular andDevelopmental Biology 33:737-741. In 2007, exosomes were discovered tobe natural carriers of RNA and protein, including over 1,300 types ofmRNA and 121 types of non-coding microRNA. Exosomes can also transmitmRNA between species: exposure of human cells to mouse exosomes carryingmouse mRNA results in translation in the human cells of the mouse mRNA.

As delivery vehicles for RNA, protein, or DNA, exosomes have a number ofadvantages over alternative vehicles. Specifically, exosomes can begenerated from a patient's own cells, making them non-immunogenic—theyare therefore not attacked by antibodies, complement, coagulationfactors, or opsonins. In addition, exosomes can be loaded with nucleicacids by electroporation, and they are naturally-occurring vehicles thatcarry mRNA and protein between human cells. Exosomes protect their RNAand protein cargo during transport, and the cargo is delivered directlyinto the cytosol. They can extravasate from the blood stream toextravascular tissues, even crossing the blood-brain barrier, and theycan be targeted. Furthermore, exosomes avoid being accumulated inuntargeted organs, such as, for example, liver. Exosomes may thereforebe used as cell-derived “liposomes” to deliver therapeutic mRNA or othercargo in the treatment of disease. Mizrak et al. (2013) MolecularTherapy 21:101-108; doi:10.1038/mt.2012.161. A graphic illustration ofan exosome delivering an mRNA to a cell is shown in FIG. 2. See also vanden Boom et al. (2011) Nature Biotechnology 29:325-326.

Most cell types are believed to be capable of generating exosomes, andexosomes are found in most biological fluids including blood, saliva,urine, cerebrospinal fluid, breast milk, and amniotic fluid. Exosomesare produced by most cell types, in different abundance. Abundantexosomes, devoid of T-cell activators, can be derived from immaturedendritic cells, which are present in human blood. O'Doherty et al.(1994) Immunology 82:487-493. Exosomes may also be producedartificially, for example by combining recombinant exosomal proteinswith lipids and phospholipids such as are found in exosomal membranes.Alternatively, exosomes may be constructed by in vitro self-assembly ofliposomes with a subset of exosomal surface proteins.

The drug delivery potential of exosomes was first demonstrated in 2011.Alvarez-Erviti et al. (2011) Nature Biotechnology 29:341-345.Specifically, exosomes were harvested from dendritic cells engineered toexpress a Lamp2B fusion protein fused to a 28 a.a. targeting ligand fromrabies virus glycoprotein (RVG), then electroporated siRNA into theexosomes and injected the exosomes into mice immunocompatible with themice from which they obtained the dendritic cells. The exosomes werethus autologous, and did not generate an immune response, as measured byIL-6, IP-10, TNF-α, and IFN-α levels. Further, repeated doses over onemonth elicited similar responses, demonstrating that there was noadaptive immune response either.

As described above, exosomes can be autologous and thus have lowimmunogenicity. Since modRNA also has low immunogenicity, thecombination of modRNA as the ribonucleic acid and an exosome as thedelivery vehicle in the compositions of the instant disclosure isparticularly preferred. In these embodiments, the disclosure thusprovides a new way of delivering mRNA or modRNA to cells or tissues,using exosomes. Such delivery provides a useful method to temporarilyincrease the level of any protein in a cell in vivo using RNA deliveredin exosomes by intravenous or topical injection, and particularly in thedelivery of an RNA encoding TERT. Accordingly, in preferred embodiments,the delivery vehicles of the instant compositions are non-immunogenic.Under some circumstances, however, it may be desirable for the vehicleto retain some immunogenicity.

Additional Components

The compositions disclosed herein may further comprise additionalcomponents that either enhance the delivery of the composition to thetarget cell, enhance the extension of telomeres within the cell, orboth. For example, the compositions may further comprise one or more ofthe compounds and conditions of Table 2. As would be understood by oneof ordinary skill in the art, combinations of active ingredients oftendisplay synergistic effects on a desired activity, such as, for example,the transient expression of exogenous telomerase activity in a cell, andsuch combinations are understood to fall within the scope of theinvention. Additional examples of proteins that may be included withinthe compositions of the instant disclosure are listed in Table 5. Itshould be understood that the compositions could either include theproteins themselves, or nucleic acid sequences, preferably RNAs ormodRNAs, that encode these proteins, or proteins with high sequenceidentity that retain the activity of the listed protein.

TABLE 5 Proteins usefully delivered in combination with TERT SpeciesProtein Activity Advantages Reference Human UPF1 Sustains telomereleading Increased rate Chawla et al. strand-replication or amount of(2011) The EMBO telomere Journal 30: 4047- extension. 4058 Human HSP90Prevents dephosphorylation of Increased Haendeler et al. Akt kinase byPP2A. Akt needs TERT activity. (2003) FEBS to be phosphorylated toLetters 536: 180- phosphorylate TERT. Also 186; Büchner et complexeswith TERT and al. (2010) keeps TERT serine 823 Antioxidants &phosphorylated, keeping TERT Redox Signaling activated. Büchner et al.(2010) 13:551-558 Antioxidants & Redox Signaling 13: 551-558. Human Aktkinase Complexes with TERT and Increased Wojtyla et al. (aka proteinHSP90, phosphorylates TERT at TERT activity. (2011) Molecular kinase B)serine 823, increasing TERT Biology Reports activity. Büchner et al.(2010) 38: 3339-3349; Antioxidants & Redox Signaling Büchner et al. 13:551-558 (2010) Antioxidants & Redox Signaling 13: 551-558 Human Proteinkinase Phosphorylates TERT, allowing Nuclear Wojtyla et al. C (PKC) (itsit to bind nuclear translocator. translocation. (2011) Molecular variousBiology Reports isoenzymes) 38: 3339-3349 Human Shp-2 Inhibitsphosphorylation of Nuclear Wojtyla et al. TERT Y707 by Src1, keepingtranslocation. (2011) Molecular TERT in nucleus. Transport of BiologyReports TERT to nucleus. 38: 3339-3349 Human TPP1 Recruits telomerase tothe Abreu et al. telomere. (2010) Molecular and Cellular Biology 30:2971- 2982 Human NFkB p65 Transport of TERT to nucleus. Nuclear Wojtylaet al. translocation. (2011) Molecular Biology Reports 38: 3339-3349Human Rap1 regulator of telomere length. Extension of O'Connor et al.telomeres. (2004) The Journal of Biological Chemistry 279: 28585-28591

Other examples of agents that may usefully be included within thecompositions of the instant disclosure are listed in Table 6.

TABLE 6 Other agents usefully delivered in combination with TERTMolecule Activity Advantages Reference Okadaic Inhibits PP2A. IncreasedWojtyla et al. (2011) Molecular acid PP2A telomerase activity BiologyReports 38: 3339-3349 dephosphorylates due to TERT AKT and or TERT.phosphorylation. AKT phosphorylates TERT, activating it. TERRA or TERRAinhibits Antisense TERRA, Cifuentes-Rojas and Shippen (2012) anti-sensetelomerase by or ARRET, should Mutat. Res. 730: 20-27; TERRA binding toTERC, increase telomerase doi:10.1016/j.mrfmmm.2011.10.003 (ARRET) towhich it is activity by binding complementary. to TERRA, preventing itfrom binding to TERC. TERC RNA component of Increase RNA telomerase,telomerase activity. essential for its function, may be second-mostlimiting factor after TERT in most cells.

Since TERT is most active during certain phases of the cell cycle, thecompositions of the instant disclosure may also optionally include oneor more transient activators of cellular proliferation, in order toenhance the effectiveness of the TERT treatment. Such agents mayinclude, for example, an RNAi agent that transiently reduces the amountsof cell cycle inhibitors such as Rb or P19/Arf in the cell. Othertransient activators of cellular proliferation may be usefully includedin the instant compositions, as would be understood by one of ordinaryskill in the art.

Methods of Extending Telomeres and Methods of Treatment

In another aspect, the instant disclosure provides methods of extendingtelomeres, comprising the step of administering any of theabove-described compounds or compositions to a cell with shortenedtelomeres, wherein telomeres are extended within the cell. The instantdisclosure also provides methods of treatment, comprising the step ofadministering any of the above-described compounds or compositions to ananimal subject in need of, or that may benefit from, telomere extension.

In preferred embodiments, the compounds or compositions are administeredto a cell, wherein the cell is an isolated cell or is part of a cellculture, an isolated tissue culture, an isolated organ, or the like(i.e., the administration is in vitro).

In other preferred embodiments, the compounds or compositions areadministered without isolating the cell or cells, the tissue, or theorgan from the subject (i.e., the administration is in vivo). In some ofthese embodiments, the compound or composition is delivered to all, oralmost all, cells in the subject's body. In some embodiments, thecompound or composition is delivered to a specific cell or tissue in thesubject's body.

In some embodiments, the subject is a mammal, bird, fish, or reptile. Inpreferred embodiments, the subject is a mammal and more preferably ahuman. In other preferred embodiments, the subject is a pet animal, azoo animal, a livestock animal, or an endangered species animal.Examples of preferred subject species are listed in Table 7.

TABLE 7 Subject animal species. Dog (Canis lupus familiaris) Sheep (Ovisaries) Domestic pig (Sus scrofa domesticus) Domestic goat (Capraaegagrus hircus) Cattle (Bos primigenius taurus) Zebu (Bos primigeniusindicus) Cat (Felis catus) Chicken (Gallus gallus domesticus) Guinea pig(Cavia porcellus) Donkey (Equus africanus asinus) Domesticated duck(Anas platyrhynchos domesticus) Water buffalo (Bubalus bubalis) Horse(Equus ferus caballus) Domesticated Silkmoth (Bombyx mori) DomesticPigeon (Columba livia domestica) Domestic goose (Anser anser domesticus)Llama (Lama glama) Alpaca (Vicugna pacos) Domesticated guineafowl(Numida meleagris) Ferret (Mustela putorius furo) Ringneck dove(Streptopelia risoria) Bali cattle (Bos javanicus domestica) Gayal (Bosfrontalis) Domesticated turkey (Meleagris gallopavo) Goldfish (Carassiusauratus auratus) Domestic rabbit (Oryctolagus cuniculus) Domestic Canary(Serinus canaria domestica) Carabao (Bubalus bubalis carabenesis)Siamese fighting fish (Betta splendens) Koi (Cyprinus carpiohaematopterus) Domesticated silver fox (Vulpes vulpes) Domesticatedhedgehog (Atelerix albiventris) Society Finch (Lonchura striatadomestica) Yak (Bos grunniens) Fancy rat and Lab rat DomesticatedDromedary Camel (Camelus dromedarius) Domesticated Bactrian Camel(Camelus bactrianus) Guppy (Poecilia reticulata some strains) Fancymouse

For in vitro applications, the compounds or compositions may beadministered using any suitable technique, as would be understood bythose skilled in the fields of cell biology, cell culture, tissueculture, organ culture, or the like. For in vivo applications, thecompounds or compositions are usefully administered by injection,topical application, inhalation, or any other suitable administrationtechnique, as would be understood by those of ordinary skill in themedical arts or the like.

As described above, cells usefully treated according to the methods ofthe disclosure include cells, either in a subject (for in vivoadministration) or from a subject (for in vitro administration), thatmay benefit from telomere extension. Since short telomeres affect almostall cell types in most animals, telomere extension may benefit mostanimals. A telomere extension treatment that is transient and brief hasthe potential to be safe, as described above. Telomere extension using atransient treatment as disclosed herein may therefore be of use in allor most individuals either as a preventive measure, for example toprevent or delay onset of the many diseases and conditions in whichshort telomeres are implicated, or as a treatment for those diseases andconditions. The treatment may benefit subjects at risk of age-relateddiseases or conditions, or who are already suffering from such diseases,and may also benefit subjects who have experienced, are experiencing, orare at risk of experiencing physical trauma or chronic physical stresssuch as hard exercise or manual labor, or psychological trauma orchronic psychological stress, since all of these conditions causetelomere shortening; physical stress or trauma requires cell division inorder to repair the resultant damage, thus shortening telomeres, andthese conditions may also cause oxidative stress, which also shortenstelomeres. Such diseases and conditions include, for example, metabolicsyndrome, diabetes, diabetic ulcers, heart disease, many forms ofcancer, vascular dementia, Alzheimer's, stroke, age-related maculardegeneration, immunosenescence, bone marrow failure, gastrointestinalulcers, cirrhosis, hernia, infection such as pneumonia secondary toimpaired immune function, chronic infection, mild or severe cognitiveimpairment, impaired mobility, osteoporosis, osteoarthritis, rheumatoidarthritis, age-related anxiety, balance disorders, tinnitus, Bell'spalsy, cataracts, COPD, corneal abrasion, coronary artery disease,peripheral artery disease, conjunctivitis, chalazion, dehydration,depression, emphysema, various eye diseases, failure to thrive, flu,generalized anxiety disorder, glaucoma, hearing loss, loss of sense oftaste, loss of appetite, hip dislocation, memory loss, Parkinson'sdisease, spinal stenosis, urinary incontinence, vertebral fracture, andothers.

Cells usefully treated according to the instant methods may also includecells in or from a subject that suffers from an age-related illness orcondition or that is at risk of suffering from such an illness orcondition. Such age-related illnesses and conditions may include geneticdiseases in which genes encoding components of the telomerase complex ortelomere complex are mutated, thus leading to short telomeres. Suchdiseases include, for example, forms of idiopathic pulmonary fibrosis,dyskeratosis congenita, and aplastic anemia.

In the case of genetic diseases, the compositions may therefore furtherinclude additional nucleic acids, such as the normal, functionalversions of the coding sequences of the genes that are affected by suchdiseases, for example DKC1, TINF2, NOP10, NHP2, TERC, or other genes.Examples of such genes are listed in Table 8. See also Armanios (2009)Ann. Rev. Genomics Hum. Genet. 10: 45-61.

TABLE 8 Genes related to age-related diseases. Age of onset in GeneDiagnosis years (typical) hTR Sporadic IPF 1-3% Broad range of hTERTFamilial IPF 8-15% ages 5-77 Sporadic and familial aplastic anemia ~3-5%Autosomal dominant dyskeratosis congenital (DC) DKC1 X-linked DC Lessthan 30 Hoyeraal-Hreiderasson Less than 5 TINF2 Sporadic DC Less than 10Autosomal dominant DC — Hoyeraal-Hreiderasson Less than 5 NOP10Autosomal Recessive DC — NHP2 Autosomal Recessive DC —

In addition, the treatment may benefit subjects suffering from, or atrisk of, other types of genetic diseases in which short telomeres mayplay a role, such as, for example, muscular dystrophy. In such diseases,the need for cell replication to address the problem caused by thegenetic mutation shortens telomeres more rapidly than normal, resultingin more rapid telomere shortening than normal, which in turn exhauststhe replicative capacity of cells, leading to tissue dysfunction,exacerbated or additional symptoms, disability, and often death. Inaddition, various types of cancer may be prevented or delayed bytreatment with compounds of the invention, and indeedchromosome-chromosome fusions caused by critically short telomeres arebelieved to be a cause of cancer. Telomeres may also be selectivelylengthened in healthy cells in an individual, while not lengtheningtelomeres in cancer cells, which may allow the instant compounds,compositions, and methods to be used, for example, to lengthen telomeresof the immune system to increase its ability to fight a cancer. Further,immune system cells may be harvested from an individual for treatmentusing the invention ex vivo followed by reintroduction into theindividual.

In some embodiments, the cells treated according to the instant methodsare from subjects where no disease state is yet manifested but where thesubject is at risk for a condition or disease involving short telomeres,or where the cells contain shortened telomeres. In some embodiments, theage-related illness is simply old age. The instant treatments may alsobe used as a cosmetic aid, to prevent, delay, or ameliorate age-relateddeterioration in appearance of skin, hair, bone structure, posture, eyeclarity, or other traits that decline with aging. For example, thetreatments may be used to help maintain skin elasticity, thickness,smoothness, and appearance, since telomere extension improves theseparameters. In cases of physical trauma such as a bone fracture or atissue crush or cut injury or burn, the invention may be used toincrease the lengths of telomeres in cells which participate in healingthe trauma, to increase their replicative capacity. In cases of chronicphysical stress, which causes telomere shortening, treatment with theinvention may lengthen telomeres in affected cells increasing theirreplicative capacity and ability to repair tissue damage. Since telomereshortening accumulates over generations, for example in humans withhaploinsufficiency of telomerase components such as hTR or hTERT,Armanios (2009) Annu. Rev. Genomics Hum. Genet. 10:45-61, the treatmentsof the instant disclosure may be applied to germ line cells such aseggs, sperm, or their precursors, or to fertilized eggs or embryos, forexample during in vitro fertilization procedures. The treatments mayalso be useful for aiding other treatments of various diseases orconditions, for example, transdifferentiation of cells in vivo. Thetreatment methods may also be useful in advance of or during surgery orchemotherapy, or radiotherapy, to increase the ability of cells toreplicate to repair damage resulting from these procedures.

The methods may also be useful for treating cells in vitro for variousapplications, including autologous or heterologous cell therapy,bioengineering, tissue engineering, growth of artificial organs,generation of induced pluripotent stem cells (iPSC), and cellulardifferentiation, dedifferentiation, or transdifferentiation. In theseapplications, cells may be required to divide many times, which may leadto loss of telomere length, which may be counteracted by the inventionbefore, during, or after the application.

In addition, various types of cancer may be considered age-relatedillnesses, particularly where the cancerous cells contain shorttelomeres. In some cases, the cells treated according to the instantmethods are from subjects where no disease state is yet manifested butwhere the cells contain shortened telomeres. In some embodiments, theage-related illness is simply the altered form and function typicallyassociated with old chronological age in humans.

The cells usefully administered compounds or compositions of the instantdisclosure according to the instant methods include cells from anytissue or cell type that may suffer the effects of shortened telomeresor that may in any way benefit from lengthening of the cell's telomeres.Cells may include somatic cells or germ cells, as well as stem cells andother progenitor cells and/or undifferentiated cells. Cells may includetumor cells and non-tumor cells.

Examples of cells usefully administered compounds or compositionsaccording to the instant methods include cells that are derivedprimarily from endoderm, cells that are derived primarily from ectoderm,and cells that are derived primarily from mesoderm. Cells derivedprimarily from the endoderm include, for example, exocrine secretoryepithelial cells and hormone-secreting cells. Cells derived primarilyfrom the ectoderm include, for example, cells of the integumentarysystem (e.g., keratinizing epithelial cells and wet stratified barrierepithelial cells) and the nervous system (e.g., sensory transducercells, autonomic neuron cells, sense organ and peripheral neuronsupporting cells, central nervous system neurons and glial cells, andlens cells). Cells derived primarily from the mesoderm include, forexample, metabolism and storage cells, barrier-function cells (e.g.,cells of the lung, gut, exocrine glands, and urogenital tract),extracellular matrix cells, contractile cells, blood and immune systemcells, germ cells, nurse cells, and interstitial cells. Accordingly, insome embodiments of the instant methods, the cell administered thecomposition is a somatic cell of endodermal, mesodermal, or ectodermallineage. In some embodiments the cell is a germ line cell or anembryonic cell.

Specific examples of cells that may be administered a compound orcomposition according to the instant methods include, e.g., salivarygland mucous cells, salivary gland serous cells, von Ebner's gland cellsin tongue, mammary gland cells, lacrimal gland cells, ceruminous glandcells in ear, eccrine sweat gland dark cells, eccrine sweat gland clearcells, apocrine sweat gland cells, gland of Moll cells in eyelid,sebaceous gland cells, Bowman's gland cells in nose, Brunner's glandcells in duodenum, seminal vesicle cells, prostate gland cells,bulbourethral gland cells, Bartholin's gland cells, gland of Littrecells, uterus endometrium cells, isolated goblet cells of therespiratory and digestive tracts, stomach lining mucous cells, gastricgland zymogenic cells, gastric gland oxyntic cells, pancreatic acinarcells, paneth cells of the small intestine, type II pneumocytes of thelung, clara cells of the lung, anterior pituitary cells (e.g.,somatotropes, lactotropes, thyrotropes, gonadotropes, andcorticotropes), intermediate pituitary cells (e.g., those secretingmelanocyte-stimulating hormone), magnocellular neurosecretory cells(e.g., those secreting oxytocin or vasopressin), gut and respiratorytract cells, (e.g., those secreting serotonin, endorphin, somatostatin,gastrin, secretin, cholecystokinin, insulin, glucagon, or bombesin),thyroid gland cells (e.g., thyroid epithelial cells and parafollicularcells), parathyroid gland cells (e.g., parathyroid chief cells andoxyphil cells), adrenal gland cells (e.g., chromaffin cells and cellssecreting steroid hormones such as mineralcorticoids andglucocorticoids), Leydig cells of testes, theca interna cells of theovarian follicle, corpus luteum cells of the ruptured ovarian follicle,granulosa lutein cells, theca lutein cells, juxtaglomerular cells,macula densa cells of the kidney, peripolar cells of the kidney,mesangial cells of the kidney, epidermal keratinocytes, epidermal basalcells, keratinocytes of fingernails and toenails, nail bed basal cells,medullary hair shaft cells, cortical hair shaft cells, cuticular hairshaft cells, cuticular hair root sheath cells, hair root sheath cells ofHuxley's layer, hair root sheath cells of Henle's layer, external hairroot sheath cells, hair matrix cells, surface epithelial cells of thestratified squamous epithelium of the cornea, tongue, oral cavity,esophagus, anal canal, distal urethra, and vagina, basal cells of theepithelia of the cornea, tongue, oral cavity, esophagus, anal canal,distal urethra, and vagina, urinary epithelium cells (e.g., lining theurinary bladder and urinary ducts), auditory inner hair cells of theorgan of Corti, auditory outer hair cells of the organ of Corti, basalcells of the olfactory epithelium, cold-sensitive primary sensoryneurons, heat-sensitive primary sensory neurons, Merkel cells of theepidermis, olfactory receptor neurons, pain-sensitive primary sensoryneurons, photoreceptor cells of the retina in the eye (e.g.,photoreceptor rod cells, photoreceptor blue-sensitive cone cells,photoreceptor green-sensitive cone cells, and photoreceptorred-sensitive cone cells), proprioceptive primary sensory neurons,touch-sensitive primary sensory neurons, type I and II carotid bodycells, type I and II hair cells of the vestibular apparatus of the ear,type I taste bud cells, cholinergic neural cells, adrenergic neuralcells, peptidergic neural cells, inner and outer pillar cells of theorgan of Corti, inner and outer phalangeal cells of the organ of Corti,border cells of the organ of Corti, Hensen cells of the organ of Corti,vestibular apparatus supporting cells, taste bud supporting cells,olfactory epithelium supporting cells, Schwann cells, satellite glialcells, enteric glial cells, astrocytes, neuron cells, oligodendrocytes,spindle neurons, anterior lens epithelial cells, crystallin-containinglens fiber cells, hepatocytes, adipocytes (e.g., white fat cells andbrown fat cells), liver lipocytes, kidney parietal cells, kidneyglomerulus podocytes, kidney proximal tubule brush border cells, loop ofHenle thin segment cells, kidney distal tubule cells, kidney collectingduct cells, type I pneumocytes, pancreatic duct cells, nonstriated ductcells (e.g., principal cells and intercalated cells), duct cells (ofseminal vesicle, prostate gland, etc.), intestinal brush border cells(with microvilli), exocrine gland striated duct cells, gall bladderepithelial cells, ductulus efferens nonciliated cells, epididymalprincipal cells, epididymal basal cells, ameloblast epithelial cells,planum semilunatum epithelial cells of the vestibular apparatus of theear, organ of Corti interdental epithelial cells, loose connectivetissue fibroblasts, corneal fibroblasts (corneal keratocytes), tendonfibroblasts, bone marrow reticular tissue fibroblasts, othernonepithelial fibroblasts, pericytes, nucleus pulposus cells of theintervertebral disc, cementoblasts/cementocytes,odontoblasts/odontocytes, hyaline cartilage chondrocytes, fibrocartilagechondrocytes, elastic cartilage chondrocytes, osteoblasts/osteocytes,osteoprogenitor cells, hyalocytes of the vitreous body of the eye,stellate cells of the perilymphatic space of the ear, hepatic stellatecells (Ito cells), pancreatic stelle cells, skeletal muscle cells (e.g.,red skeletal muscle cells (slow) and white skeletal muscle cells(fast)), intermediate skeletal muscle cells, nuclear bag cells of themuscle spindle, nuclear chain cells of the muscle spindle, satellitecells, heart muscle cells (e.g., ordinary heart muscle cells, nodalheart muscle cells, and Purkinje fiber cells, smooth muscle cells(various types), myoepithelial cells of the iris, myoepithelial cells ofthe exocrine glands, erythrocytes, megakaryocytes, monocytes, connectivetissue macrophages (various types). epidermal Langerhans cells,osteoclasts, dendritic cells, microglial cells, neutrophil granulocytes,eosinophil granulocytes, basophil granulocytes, hybridoma cells, mastcells, helper T cells, suppressor T cells, cytotoxic T cells, naturalkiller T cells, B cells, natural killer cells, reticulocytes, stem cellsand committed progenitors for the blood and immune system (varioustypes), oogonia/oocytes, spermatids, spermatocytes, spermatogoniumcells, spermatozoa, nurse cells, ovarian follicle cells, sertoli cells,thymus epithelial cells, and interstitial kidney cells.

In a preferred embodiment, the cells administered a compound orcomposition according to the instant methods are stem or progenitorcells, since these cells give rise to other cells of the body. Inanother preferred embodiment, the cells treated are cells in whichtelomeres shorten more quickly than in other cell types, for exampleendothelial cells, fibroblasts, keratinocytes, cells of the immunesystem including thyroid and parathyroid cells and leukocytes and theirprogenitors, cells of the intestines, liver, mucosal membrane cells,e.g. in the esophagus and colon, and cells of the gums and dental pulp.

Accordingly, in some embodiments, the cells are fibroblast cells,keratinocytes, endothelial cells, epithelial cells, or blood cells.

The administering step may be performed one or more times depending onthe amount of telomere extension desired. In some embodiments of theinstant methods, the cell is an isolated cell, and the administeringstep lasts no longer than 96 hours, no longer than 72 hours, no longerthan 48 hours, no longer than 36 hours, no longer than 24 hours, nolonger than 18 hours, no longer than 12 hours, no longer than 8 hours,no longer than 4 hours, or even shorter times. In some embodiments, theadministering step lasts at least 2 hours, at least 4 hours, at least 8hours, at least 12 hours, at least 18 hours, at least 24 hours, at least36 hours, at least 48 hours, or even longer times. In preferredembodiments, the administering step lasts no longer than 48 hours, nolonger than 96 hours, or no longer than 1 week. In other preferredembodiments, the administering step lasts at least 2 hours. It should beunderstood that, in the case where administration is by transfection,the time for administration includes the time for the cell to recoverfrom the transfection method.

In some embodiments of the instant methods, the cell is an isolatedcell, and the administering step is performed no more than 6 times, nomore than 5 times, no more than 4 times, no more than 3 times, no morethan 2 times, or even no more than 1 time. In some embodiments, theadministering step is performed not less than 2 times, not less than 3times, not less than 4 times, not less than 5 times, not less than 6times, or even more often.

In some embodiments, the administering step is performed once or a fewtimes over a relatively brief period to re-extend telomeres, and thennot performed for a prolonged period until telomeres need to be extendedagain. This cycle may be repeated indefinitely. Such a treatmentschedule allows telomeres to be periodically re-extended, with intervalsin between administration steps during which telomeres shorten. Periodictreatment methods may be performed either by in vivo administration orby in vitro administration, as desired. In some embodiments, theadministering step in such a series is performed no more than 6 times,no more than 5 times, no more than 4 times, no more than 3 times, nomore than 2 times, or even no more than 1 time. In some embodiments, theadministering step is performed not less than 2 times, not less than 3times, not less than 4 times, not less than 5 times, not less than 6times, or even more often. By varying the number of times theadministering step is performed, and the dose of the compounds ourcompositions of the invention used, the amount of telomere extensionachieved can be controlled.

In some embodiments, the methods of the instant disclosure furtherinclude the step of culturing the cell on a specific substrate,preferably an elastic substrate. Such substrates are known to preventunwanted changes in the cell that would normally occur on othersubstrates due to the non-physiological elasticity of those substrates.See PCT International Publication No. WO2012/009682, which isincorporated by reference herein in its entirety. Elastic substrates mayadditionally promote cell survival.

Administration of the compounds or compositions of the instantdisclosure results in the transient expression of a telomerase activityin the cell. The increased activity is readily measured by variousassays, such as, for example, the Trapeze® RT telomerase detection kit(Millipore), which provides a sensitive, real-time in vitro assay usingfluorimetric detection and quantitation of telomerase activity, althoughother measurement techniques are also possible. In some embodiments, thetelomerase activity is increased by at least 5%, at least 10%, at least20%, at least 30%, at least 50%, or even more. In preferred embodiments,the telomerase activity is increased by at least 5%.

As previously noted, one of the advantages of the instant techniques isthat the expression of telomerase activity is transient in the treatedcells. In particular, such transient expression is in contrast toprevious techniques where a telomerase reverse transcriptase gene isinserted into the genomic sequence of the cell or otherwise permanentlymodifies the genetic make-up of the targeted cell and results inconstitutive activity of the nucleic acid sequence.

FIG. 3 graphically illustrates some of the advantages of the compounds,compositions, and methods disclosed herein. In particular, the speed oftelomere extension made possible with these compounds, compositions, andmethods enables telomere maintenance by very infrequent delivery of TERTmodRNA. The expressed telomerase activity rapidly extends telomeres in abrief period, before being turned over, thus allowing the protectiveanti-cancer mechanism of telomere-shortening to function most of thetime. Between treatments, normal telomerase activity and telomereshortening is present, and therefore the anti-cancer safety mechanism oftelomere shortening to prevent out-of-control proliferation remainsintact, while the risk of short telomere-related disease remains low. Incontrast, the best existing small molecule treatment for extendingtelomeres requires chronic delivery, and thus presents a chronic cancerrisk, and even then has a small, inconsistent effect on telomere length,with no detectable effect on telomere length at all in about half ofpatients.

Accordingly, in some embodiments of the instant methods, the expressionof telomerase reverse transcriptase activity, i.e., the half-life oftelomerase activity, lasts no longer than 48 hours, no longer than 36hours, no longer than 24 hours, no longer than 18 hours, no longer than12 hours, no longer than 8 hours, no longer than 4 hours, or evenshorter times. In some embodiments, the expression of telomerase reversetranscriptase activity lasts at least 2 hours, at least 4 hours, atleast 8 hours, at least 12 hours, at least 18 hours, at least 24 hours,at least 36 hours, at least 48 hours, or even longer times. In preferredembodiments, the expression of telomerase reverse transcriptase activitylasts no longer than 48 hours. In other preferred embodiments, theexpression of telomerase reverse transcriptase activity lasts at least 2hours.

In some embodiments of the instant methods, the transient expression isindependent of cell cycle.

As noted above, the transient expression of telomerase reversetranscriptase results in the extension of shortened telomeres in treatedcells. Telomere length can be readily measured using techniques such asterminal restriction fragment (TRF) length analysis, qPCR, MMqPCR, andQ-FISH, as would be understood by one of ordinary skill in the art. See,e.g., Kimura et al. (2010) Nat Protoc. 5:1596-607; doi:10.1038/nprot.2010.124. In some embodiments, the instant methodsincrease telomere length in treated cells by at least 0.1 kb, at least0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, or evenmore.

One of the advantages of the instant compounds, compositions, andmethods, is the rapidity of extension of telomeres achieved by thesetechniques. The techniques allow treatments to be brief and thus safebecause the normal protective telomere shortening mechanism remainsintact for most of the time. Treatment with the compounds andcompositions disclosed herein result in delivery of tens or hundreds ofcopies of TERT modRNA per cell as measured by absolute RT-qPCR, which issubstantially more than the average number of copies of endogenous TERTmRNA found even in cells with high telomerase activity. Typically suchcells have less than one copy of TERT mRNA per cell (Yi et al. (2001)Nucl. Acids Res. 29:4818-4825). Thus the treatments transientlyintroduce a large number of copies of modRNA encoding TERT to a cellresulting in rapid telomere extension. Without intending to be bound bytheory, the large number of copies of modRNA encoding TERT maytransiently overwhelm the inhibitory regulatory mechanisms that normallyprevent TERT, and other methods of telomere extension, from extendingtelomeres as rapidly as the compounds, compositions, and methodsdisclosed herein.

The transient expression of telomerase reverse transcriptase alsoresults in an increased replicative capacity in treated cells. Increasedreplicative capacity is readily monitored in cells that are approachingreplicative senescence by measuring additional population doublings insuch cells. Senescent cells are not stimulated to divide by passage inculture or treatment with serum. Senescent cells are further oftencharacterized by the expression of pH-dependent β-galactosidaseactivity, expression of cell cycle inhibitors p53 and p19, and otheraltered patterns of gene expression, and an enlarged cell size. Absenttreatment with the compounds and compositions of the instant disclosure,human lung fibroblast cells typically double 50-60 times. With one setof one to three treatments lasting only a few days total, however, thesecells achieve an additional 16-28 population doublings. If treated againseveral weeks later, additional proliferative capacity is conferredagain. This process of intermittent treatments to periodically re-extendtelomeres may be applied additional times, with the interval betweentreatments depending on factors such as the rate of telomere shortening,the rate of cell divisions, and the amount of telomere extensionprovided by the treatment. Likewise, human microvascular dermalendothelial cells from an aged individual, absent treatment with theinstant compositions, may achieve only 1-2 population doublings, whereastreated cells may achieve 3, 4, or even more population doublings.

Accordingly, in some embodiments, the instant treatment methods increasethe number of population doublings by at least one, two, four, or evenmore population doublings. In some embodiments, the treatment methodsincrease the number of population doublings by at least 5, 10, 15, 20,or even more population doublings.

In some of the instant method embodiments, the compounds or compositionsof the invention are administered to the animal cell by electroporation.In specific embodiments, a compound of the invention is administered tothe animal cell by electroporation in the absence of a delivery vehicle.In other specific embodiments a compound of the invention and atelomerase RNA component are administered to the animal cell byelectroporation.

Kits

In another aspect, the instant disclosure provides ready-to-use kits foruse in extending telomeres in a mammalian cell. The kits comprise any ofthe above-described compounds or compositions, together withinstructions for their use. In some embodiments, the kits furthercomprise packaging materials. In preferred embodiments, the packagingmaterials are air-tight. In these embodiments, the packaging materialsmay optionally be filled with an inert gas, such as, for example,nitrogen, argon, or the like. In some embodiments, the packagingmaterials comprise a metal foil container, such as, for example, asealed aluminum pouch or the like. Such packaging materials are wellknown by those of ordinary skill in the art.

In some embodiments, the kit may further comprise a desiccant, a culturemedium, an RNase inhibitor, or other such components. In someembodiments, the kit may further comprise a combination of more than oneof these additional components. In some kit embodiments, the compositionof the kit is sterile.

Further Aspects

In yet another aspect, the invention provides novel compounds,compositions, kits, and methods according to the following numberedparagraphs:

1. A composition for the extension of telomeres comprising:

a ribonucleic acid comprising at least one modified nucleoside andcoding for a telomerase reverse transcriptase; and

a delivery vehicle for the ribonucleic acid;

wherein telomeres are extended within a cell treated with thecomposition.

2. The composition of paragraph 1, wherein the telomerase reversetranscriptase is a mammalian, avian, reptilian, or fish telomerasereverse transcriptase or a variant that retains telomerase catalyticactivity.

3. The composition of paragraph 2, wherein the telomerase reversetranscriptase is a human telomerase reverse transcriptase.

4. The composition of paragraph 1, wherein the ribonucleic acidcomprises a 5′ cap, a 5′ untranslated region, a 3′ untranslated region,and a poly-A tail.

5. The composition of paragraph 4, wherein the poly-A tail increasesstability of the ribonucleic acid.

6. The composition of paragraph 4, wherein the 5′ untranslated region orthe 3′ untranslated region comprise a sequence from a stable mRNA or anmRNA that is efficiently translated.

7. The composition of paragraph 4, wherein the 5′ untranslated regionand the 3′ untranslated region both comprise a sequence from a stablemRNA or an mRNA that is efficiently translated.

8. The composition of paragraph 4, wherein the 5′ cap, the 5′untranslated region, or the 3′ untranslated region stabilizes theribonucleic acid, increases the rate of translation of the ribonucleicacid, or reduces the immunogenicity of the ribonucleic acid.9. The composition of paragraph 1, wherein the at least one modifiednucleoside reduces immunogenicity of the ribonucleic acid.10. The composition of paragraph 1, wherein the ribonucleic acid is asynthetic ribonucleic acid.11. The composition of paragraph 10, wherein the synthetic ribonucleicacid is a purified synthetic ribonucleic acid.12. The composition of paragraph 11, wherein the synthetic ribonucleicacid is purified to remove immunogenic components.13. The composition of paragraph 1, wherein the ribonucleic acid codesfor a human, cat, dog, mouse, cow, sheep, pig, African elephant,chicken, rat, zebrafish, Japanese medaka, or chimpanzee telomerasereverse transcriptase, or a polypeptide with at least 95% sequenceidentity to the telomerase reverse transcriptase.14. The composition of paragraph 1, wherein the composition furthercomprises a telomerase RNA component.15. The composition of paragraph 14, wherein the telomerase RNAcomponent is a mammalian, avian, reptilian, or fish telomerase RNAcomponent.16. The composition of paragraph 15, wherein the telomerase RNAcomponent is a human telomerase RNA component.17. The composition of paragraph 1, wherein the delivery vehicle is anexosome, a lipid nanoparticle, a polymeric nanoparticle, a natural orartificial lipoprotein particle, a cationic lipid, a protein, aprotein-nucleic acid complex, a liposome, a virosome, or a polymer.18. The composition of paragraph 17, wherein the delivery vehicle is acationic lipid.19. The composition of paragraph 1, wherein the delivery vehicle isnon-immunogenic.20. A method of extending telomeres, comprising the step of:

administering the composition of any one of paragraphs 1 to 19 to ananimal cell, wherein at least one telomere is extended within the cell.

21. The method of paragraph 20, wherein the cell has at least oneshortened telomere prior to the administering step.

22. The method of paragraph 20, wherein the cell is from a subjectsuffering from or at risk of an age-related illness, an age-relatedcondition, or an age-related decline in function or appearance.

23. The method of paragraph 20, wherein the cell is from a subjectsuffering from or at risk of cancer, heart disease, stroke, diabetes,Alzheimer's disease, osteoporosis, a decline in physical ability orappearance, physical trauma or chronic physical stress, or psychologicaltrauma or chronic psychological stress.24. The method of paragraph 20, wherein the cell is a somatic cell ofendodermal, mesodermal, or ectodermal lineage, or a germ line orembryonic cell.25. The method of paragraph 20, wherein the cell is an inducedpluripotent stem cell or a cell used to produce an induced pluripotentstem cell.26. The method of paragraph 20, wherein the cell is atransdifferentiated cell or a cell used to produce a transdifferentiatedcell.27. The method of paragraph 20, wherein the cell is an isolated cell,and the administering step lasts no longer than 48 hours.28. The method of paragraph 20, wherein the cell is an isolated cell,and the administering step is performed no more than four times.29. The method of paragraph 20, wherein the cell is an isolated cell,and the method further comprises the step of measuring telomeraseactivity in the cell.30. The method of paragraph 29, wherein the administering step increasestelomerase activity in the cell.31. The method of paragraph 30, wherein the telomerase activity istransiently increased by at least 5%.32. The method of paragraph 30, wherein the half-life of increasedtelomerase activity lasts no longer than 48 hours.33. The method of paragraph 20, wherein the cell is an isolated cell,and the method further comprises the step of measuring average telomerelength in the cell.34. The method of paragraph 33, wherein average telomere length isincreased by at least 0.1 kb.35. The method of paragraph 20, wherein the cell is an isolated cell,and the method further comprises the step of measuring populationdoubling capacity in the cell.36. The method of paragraph 35, wherein the population doubling capacityincreases by at least 25%.37. The method of paragraph 20, wherein the cell is from a mammaliansubject.38. The method of paragraph 37, wherein the cell is from a humansubject.39. The method of paragraph 20, wherein the cell is an isolated cell.40. The method of paragraph 20, wherein the cell is not an isolatedcell.41. A kit for extending telomeres in an animal cell, comprising:

the composition of any one of paragraphs 1 to 19; and

instructions for using the composition to extend telomeres.

42. The kit of paragraph 41, further comprising packaging materials.

43. The kit of paragraph 42, wherein the packaging materials areair-tight.

44. The kit of paragraph 42, wherein the packaging materials comprise ametal foil container.

45. The kit of paragraph 41, further comprising a desiccant.

46. The kit of paragraph 41, further comprising a culture medium.

47. The kit of paragraph 41, further comprising an RNase inhibitor.

48. The kit of paragraph 41, wherein the composition is sterile.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the compounds,compositions, methods, and kits described herein may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following Examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Example 1. Highly Efficient Telomere Extension in Human CellsUsing Modified mRNA Encoding Telomerase

Diseases of inadequate telomere length maintenance and the need forincreased cell replicative capacity for cell therapies andbioengineering applications motivate development of safe methods fortelomere extension. Blackburn et al. (2010) Cancer Prev Res (Phila)3:394-402; Calado et al. (2012) Leukemia 26:700-707; Alter et al. (2009)Blood 113:6549-6557; Mohsin et al. (2012) Journal of the AmericanCollege of Cardiology doi:10.1016/j.jacc.2012.04.047. mRNA delivery totransiently increase the amount of protein encoded by the mRNA fortherapeutic applications is facilitated by incorporation of modifiednucleosides reduce immunogenicity and increase stability. Karikó et al.(2005) Immunity 23:165-175; Karikó et al. (2011) Nucleic Acids Res.39:e142; doi: 10.1093/nar/gkr695.

To be therapeutically useful, a telomere extension treatment shouldideally be non-immunogenic; specific; capable of being initiated beforetelomeres shorten to the critically short lengths that cause chromosomalinstability and increased cancer risk (Wentzensen et al. (2011) CancerEpidemiol. Biomarkers Prev. 20:1238-1250; Calado et al. (2012) Leukemia26:700-707; Artandi and DePinho (2010) Carcinogenesis 31:9-18);transient and intermittent, to allow the normal anti-cancer telomereshortening mechanism to function almost continuously; effective even inslow-dividing cells such as some progenitor and stem cell populations;deliverable in vitro and in vivo using non-immunogenic vehicles; and,finite, enabling only enough additional cell divisions to potentiallyameliorate or prevent diseases of inadequate telomere maintenance, or toenable sufficient amplification of cells for cell therapies orbioengineering applications.

Existing treatments, including treatments using small molecules, do notmeet all of these criteria. Harley et al. (2011) Rejuvenation Res.14:45-56. Viral delivery of TERT, while possibly inducible, risksinsertional mutagenesis and thus presents serious safety concerns.Treatments involving continuous telomerase overexpression arepotentially unsafe, because in a cell with an oncogenic mutation, eitherdue to critically short telomeres and resulting chromosomal instability(O′Sullivan and Karlseder (2010) Nat. Rev. Mol. Cell Biol. 11:171-181)or another cause, constitutive telomerase expression, either due to asecond mutation or a drug, may support malignancy by enabling unlimitedproliferation. Artandi and DePinho (2010) Carcinogenesis 31:9-18; Dinget al. (2012) Cell 148:896-907.

The criteria for safe telomere extension are met by an RNA-basedapproach, facilitated by the recent discovery that certainnaturally-occurring nucleosides found in RNA increase the stability andreduce the immunogenicity of exogenous RNA when delivered to cells.Karikó et al. (2005) Immunity 23:165-175. Examples of such nucleosidesare listed in Table 4. Without intending to be bound by theory, thesemodified versions of canonical nucleosides may allow Toll-like receptorsof the innate immune system to distinguish endogenous modRNA fromforeign RNA not containing these nucleotides. Delivery of sufficientlypurified synthetic modRNA containing these non-canonical nucleosides tocells results in increased stability of the modRNA and transientelevation of the protein encoded by the modRNA with reduced or abrogatedimmune response. Karikó et al. (2011) Nucleic Acids Res. 39:e142; doi:10.1093/nar/gkr695. Thus, delivery of exogenous modRNA presentsunprecedented opportunities for applications which require transientprotein production. For example, biomimetic modRNA has been transfectedinto fibroblasts, thus reprogramming them to pluripotent stem cells.Yakubov et al. (2010) Biochem. Biophys. Res. Commun. 394:189-193.Biomimetic modRNA has also been injected into mice to rescue a model ofpulmonary surfactant protein deficiency and to elevate erythropoietinand hematocrit levels. Kormann et al. (2011) Nat. Biotechnol.29:154-157. As described above, the nucleosides used in the modRNAs usedherein were chosen not only to increase the stability but also to reduceor abrogate immunogenicity and to increase translational efficiency ofthe RNA. Transfection of dendritic cells using an unmodified mRNAencoding hTERT, while resulting in an increase in telomerase activitywithin the cells also elicited a strong hTERT-specific cytotoxic Tlymphocyte response. Saeboe-Larssen et al. (2002) Journal ofImmunological Methods 259: 191-203. Approaches using unmodified RNAs aretherefore not likely to be effective in extending telomeres in cells.

Thus, delivery of modRNA encoding TERT to cells may be used totransiently increase levels of TERT protein and telomerase activitysufficiently to extend telomeres and increase replicative capacity by afinite amount. The increased levels of telomerase activity occur rapidlyenough to enable a telomere extension treatment that is brief andinfrequent (see FIG. 1a and FIG. 3).

To demonstrate the approach, TERT modRNA was synthesized using ratios ofcanonical and non-canonical nucleosides that confer stability, efficienttranslation, and reduced or abrogated immunogenicity. Yakubov et al.(2010) Biochem. Biophys. Res. Commun. 394:189-193; Karikó et al. (2011)Nucleic Acids Res. 39:e142; doi: 10.1093/nar/gkr695. The syntheticmodRNA contains the 5′ and 3′ UTRs of beta globin mRNA, which has arelatively long half-life. To further enhance stability the modRNA has along, 151 nucleotide poly-A tail. To maximize the fidelity of the longTERT DNA template used in the in vitro transcription reaction togenerate RNA, the DNA templates were generated using a plasmid- ratherthan PCR-based approach.

To distinguish bona fide telomere extension from selection of cells withlong telomeres from a heterogeneous starting population, a controlmodRNA was synthesized that encodes a catalytically inactive form ofTERT (CI TERT) with the single residue mutation D712A, in which one ofthe triad of metal-coordinating aspartates at the catalytic site of thereverse transcriptase domain is substituted with alanine, abrogating thecatalytic activity of CI TERT but leaving it structurally intact to theextent of being able to bind template DNA, and as stable as TERT inreticulocyte lysate. Wyatt (2009) “Structure-Function Analysis of theHuman Telomerase Reverse Transcriptase” University of Calgary, Ph.D.Thesis(http://dspace.ucalgary.ca/bitstream/1880/47511/1/2009_Wyatt_PhD.pdf).

MRC-5 human fetal lung fibroblasts were chosen as test cells in thisexample because these and other human fibroblast strains have served fordecades as workhorses in the telomere field and there is thus abundantdata to inform experimental design and analysis. MRC-5 cells also haverelatively low endogenous telomerase activity, and exhibit telomereshortening and eventual senescence. Since oxidative stress increases therate of telomere shortening in MRC-5 cells (von Zglinicki et al. (2000)Free Radic. Biol. Med. 28:64-74), and causes TERT to localize to thecytoplasm where it cannot extend telomeres (Ahmed et al. (2008) J. Cell.Sci. 121:1046-1053), the cells were cultured in 5% rather than ambientoxygen. To further increase the likelihood of success the cells werealso cultured in medium optimized for protein production in MRC-5 cells(Wu et al. (2005) Cytotechnology 49:95-107).

The efficiency of modRNA-based telomere extension is subject to severalfactors, including efficiency of transfection, translation, and foldinginto functional telomerase, and ability to at least transiently escapethe extensive mechanisms of post-translational regulation that inhibitTERT and telomerase in many cell types including MRC-5 cells. While thetransfection efficiencies with smaller modRNA species such as nGFP (0.8kb) in MRC-5 cells are typically over 90% even with low concentrationsof modRNA (FIG. 6), the TERT open reading frame is relatively large(3399 bp), and the modRNA TERT construct includes UTRs and a poly-Atail, thus making the construct even larger (3751 bp). As shown in FIG.1b , however, both TERT and CI TERT were efficiently transfected intoMRC-5 cells by cationic lipid (as measured by RT-PCR of mRNA harvestedat end of treatment from MRC-5 cells treated with 1 ug/ml TERT modRNAfor 5 hours). Further, delivery of TERT or CI TERT modRNA resulted inequivalent and significant (P<0.03 and <0.01, respectively) increases inthe amount of protein recognized by anti-TERT antibody, with a size ofapproximately 122 kDa, close to the estimated size of TERT of 127 kDa(FIG. 1c ). Wick et al. (1999) Gene 232:97-106. Protein levels weremeasured by quantitative infrared fluorescence Western blot. TERTprotein levels do not differ significantly between cells treated withTERT and cells treated with CI TERT (n=3).

To form functional telomerase, TERT must fold properly and form acomplex with at least TERC. Telomerase is also heavily regulatedpost-translationally, including by cytoplasmic localization andinhibitory phosphorylation. Cifuentes-Rojas and Shippen (2012) Mutat.Res. 730:20-27; doi:10.1016/j.mrfmmm.2011.10.003. Indeed not all cellsexpressing TERT are able to maintain telomeres. Counter et al. (1998)Proc. Natl. Acad. Sci. U.S.A. 95:14723-14728. Delivery of TERT modRNAincreased telomerase activity in a dose-dependent manner up to a maximumat a concentration of approximately 0.6 ug/ml (see FIG. 7), and aconcentration of 1 ug/ml was used for subsequent experiments. Telomeraseactivity levels increased rapidly but returned to baseline levels withinapproximately 48 hours, and CI TERT modRNA caused no change intelomerase activity (FIG. 1d ). Telomerase activity was measured byqPCR-based TRAPeze® RT assay (n=3).

Since post-translational regulation of TERT is mediated in part bycytoplasmic sequestration in a cell cycle-dependent manner, thesubcellular localization of TERT in treated and untreated cells was alsoinvestigated. Cifuentes-Rojas et al. (2011) Mutat. Res.doi:10.1016/j.mrfmmm.2011.10.003. Consistent with the Western blotresults, immunocytochemistry showed abundant, though possibly inhibited,protein recognized by TERT antibody in untreated MRC-5 cells, making itdifficult to distinguish endogenous from exogenous TERT (data notshown).

Cells within approximately 10 population doublings (PD) of the onset ofreplicative senescence were used to measure the effect of TERT modRNAtreatment on telomere length and replicative capacity. The decision touse such cells was based on at least two factors. First, cells at thisstage have relatively short telomeres, and since telomerasepreferentially extends short telomeres, including in MRC-5 cells,treatment of cells at this stage should result in a larger, more easilymeasured effect. Britt-Compton et al. (2009) FEBS Lett. 583:3076-3080.Second, by treating cells at this stage, the amount of time required todetermine whether the treatment increased replicative capacity isreduced. Under the instant conditions, replicative senescence beginsapproximately 50 PD after receipt of MRC-5 cells from the supplier (seeMethods for details), and thus it was decided to treat cells atapproximately PD 40. MRC-5 cells at this stage have average telomerelengths of approximately 5-7 kb, depending on culture history and PD onreceipt from suppliers. Sitte et al. (1998) Free Radic. Biol. Med.24:885-893; MacKenzie et al. (2000) Exp. Cell Res. 259:336-350.

Several considerations were taken into account in designing thetreatment schedule (FIG. 4a ). Rates of telomere extension obtainedusing viral transduction of TERT into MRC-5 cells vary but are on theorder of about 0.2 kb per division. MacKenzie et al. (2000) Exp. CellRes. 259:336-350. Rates of telomere shortening in MRC-5 cells vary withculture conditions and practices (Sitte et al. (1998) Free Radic. Biol.Med. 24:885-893; von Zglinicki et al. (2000) Free Radic. Biol. Med.28:64-74), but are approximately 0.1 kb per PD in ambient oxygen (Sitteet al. (1998) Free Radic. Biol. Med. 24:885-893). Cells were cultured in5% oxygen, as MRC-5 cell telomeres shorten more slowly under lessoxidative conditions. von Zglinicki et al. (2000) Free Radic. Biol. Med.28:64-74. The PD time in PD 40 MRC-5 cells was found to be approximately33 hours. Given these data, it might be expected that multipletreatments would be required in order to detect telomere extensionwithin the resolution of the chosen methods of telomere lengthmeasurement, telomere restriction fragment (TRF) analysis andquantitative fluorescence in situ hybridization (Q-FISH). The intervalbetween treatments was chosen to be 48 hours since it was found thattelomerase activity levels returned to baseline levels by this timeafter a single treatment.

As shown in FIGS. 3(b)-(d), treatment of MRC-5 cells with hTERT modRNAresults in a significant increase in telomere length in these cellscompared to cells that are either untreated or treated with the deliveryvehicle only. Fluorescence micrographs of metaphase MRC-5 cellsfollowing treatment shows the location of a telomere probe on thechromosomes in these cells (FIG. 4(e)).

The effect of the treatment on telomere length was also measured byquantitative fluorescence in situ hybridization (Q-FISH) (FIG. 4(f)),because this technique provides relatively high resolution (0.3 kb) andbecause the fluorescence of Q-FISH telomere probes is directlyproportional to telomere length. Lansdorp et al. (1996) Hum. Mol. Genet.5:685-691; Martens et al. (1998) Nat. Genet. 18:76-80. Becausereplicative capacity is the functional parameter of most interest inincreasing as a result of extending telomeres, a standard curve relatingpopulation doubling number of MRC-5 cells to telomere length as measuredusing Q-FISH was constructed (FIG. 4(g)(i)). After three treatments ofPD 40 MRC-5 with TERT mRNA at 48 hour intervals, average total telomerelength per cell increased by 56+/−5% (n=15 cells for each of twobiological replicates for each treatment; error bars represent s.e.m.).Telomere lengths in treated PD 40 cells were similar to those ofuntreated PD 3 cells (FIG. 4(g)(ii)) (upper dashed line), and thus thetreatment extended telomeres by the amount by which telomere shorten inthese cells over 37 PD. In this number of PD under standard cultureconditions, MRC-5 telomeres shorten by over >1 kb. Sitte et al. (1998)Free Radic. Biol. Med. 24:885-893. Consistent with this, the observedincrease in telomere length of 56% corresponds to an increase in averagetelomere length of approximately 0.6-2.5 kb, based on the fact that PD40 MRC-5 cells have average telomere lengths of approximately 5-7 kb asmeasured using TRF (Sitte et al. (1998) Free Radic. Biol. Med.24:885-893; MacKenzie et al. (2000) Exp. Cell Res. 259:336-350), whichoverestimates telomere length by approximately 2.5-4 kb, and thus actualaverage telomere lengths in PD 40 MRC-5 cells are in the range of 1-4.5kb. Aubert et al. (2012) Mutat. Res. 730:59-67. Since the treatmentlasted a total of 144 hours, and the population doubling time of thecells was approximately 33 h. during this time, the cells underwentapproximately 4-5 PD. Thus, the rate of telomere extension wasapproximately 0.1-0.6 kb per PD, the upper range of which approaches themaximum rates ever reported following viral transduction of DNA encodingTERT, and the lower range of which is comparable to typical rates seenusing viral delivery. As expected, the untreated cells have telomerelengths equivalent to PD 40 cells during generation of the standardcurve (lower dashed line in FIG. 4(g)).

Next, the effect of TERT mRNA treatment on cell replicative capacity wasexamined. As expected, untreated and vehicle only-treated cells senescedwithin the normal range of 50-60 PD (FIG. 5a ). In striking contrast,cells treated with TERT mRNA continued to proliferate beyond the PD atwhich the control populations reached replicative senescence in adose-dependent fashion, with each additional treatment conferringsignificantly more extra PD.

Three treatments with TERT mRNA resulted in an increase in replicativecapacity of 28+/−1.5 PD. This result is consistent with an estimate thattelomeres were extended by >1 kb, as MRC-5 telomeres shorten byapproximately 0.1 kb per PD (Sitte et al. (1998) Free Radic. Biol. Med.24:885-893) in ambient (20%) oxygen, and the treated cells were culturedin 5% oxygen, in which telomeres shorten more slowly. The observedincrease in replicative capacity of treated cells of 28 PD is equivalentto the loss of replicative capacity that occurs in normal fibroblastsover more than a decade of normal human aging. Takubo et al. (2010)Geriatr Gerontol Int. 10 Suppl 1:S197-206; doi:10.1111/j.1447-0594.2010.00605.x.; Allsopp, R. C. et al. (1992) Proc.Natl. Acad. Sci. U.S.A. 89:10114-10118.

Importantly for therapeutic applications, all of the treated cellsstudied to date eventually senesced, and indeed senesced in fewer PDthan untreated cells with equivalent telomere lengths, as might beexpected since the treated cells have undergone dozens of additional PD,compared to the untreated cells with similar-length telomeres, in whichto accumulate non-telomeric DNA damage or other damage that might affectrates of telomere shortening or otherwise support induction ofreplicative senescence.

As MRC-5 cells approached and entered replicative senescence, theytended to swell to several times the diameter of early passages (FIG. 8a). As with replicative senescence, this transition was delayed in cellstreated with TERT modRNA, but not in cells receiving CI TERT modRNA orvehicle only (FIG. 8b ).

Consistent with the finding that CI TERT modRNA caused no increase intelomerase activity (FIG. 1d ), CI TERT modRNA treatment did not changetelomere length distribution relative to untreated cells, and nor did CITERT modRNA increase replicative capacity, despite causing an increasein TERT protein level equivalent to that caused by TERT modRNA (FIG. 1c). Since the only difference between TERT and CI TERT is asingle-residue substitution which abrogates the ability of CI TERT totransfer nucleotides to telomeres, these results strongly support thehypothesis that the increase in telomere length and replicative capacityobserved in cells treated with TERT modRNA is due to bona fide telomereextension in at least some of the treated cells, rather than selectionof a pre-existing subpopulation of cells with longer telomeres.

The results of this example show that delivery of TERT modRNA to humancells transiently elevates telomerase activity, rapidly extendstelomeres, and increases replicative capacity by a finite, and thuspotentially safe, amount. Purified biomimetic modRNA is alsohypo-immunogenic. Karikó et al. (2011) Nucleic Acids Res. 39:e142; doi:10.1093/nar/gkr695. Thus modRNA delivery meets several importantcriteria for a therapeutic telomere extension treatment, and also haspromise with regard to other criteria including specificity, targeteddelivery, and ability to overcome post-translational regulation.Regarding specificity, TERT overexpression may also affect other genessuch as Wnt (Park et al. (2009) Nature 460:66-72), but such effects maybe avoided by delivering modRNA encoding a TERT mutated at binding sitesof factors that mediate any such non-specific effects. Regardingdelivery, the recent discovery that in humans modRNA is transported inexosomes between cells via blood and other body fluids enablesexosome-based modRNA delivery for telomere extension and otherapplications. Lakhal and Wood (2011) Bioessays 33:737-741. Exosomes havebeen used successfully to deliver biomimetic modRNA to cells in vitro(data not shown). Regarding post-translational regulation, cellcycle-dependent and independent inhibitory post-translational regulationof TERT may be avoided by delivering modRNA encoding TERT mutated at oneor more known sites that mediate those activities, such as the nuclearexport sequence, or by co-delivering modRNA encoding other members ofthe telomerase or telomere complexes. These approaches enable telomereextension even in slowly- or non-dividing cells, making TERT modRNAtreatment appropriate for quiescent or slow-dividing stem and progenitorcell populations. modRNA-based telomere extension thus finds immediateuse in increasing the replicative capacity of cells for research, andadditionally for cell therapies, bioengineering applications, and invivo treatments that address diseases and conditions of inadequatetelomere maintenance.

In summary, modRNA encoding human telomerase reverse transcriptase(TERT) was delivered to MRC-5 human lung fibroblasts three times over 96hours. Telomeres in treated cells were extended by >1 kb, an amount bywhich fibroblast telomeres shorten over more than a decade of aging inhumans on average. Takubo et al. (2010) Geriatr Gerontol Int. 10 Suppl1:S197-206; doi: 10.1111/j.1447-0594.2010.00605.x. Telomerase activityreturned to pre-treatment levels within 48 hours and the onset ofreplicative senescence in the treated cells was delayed by approximately30 population doublings, in a dose-dependent manner. Thus delivery oftelomerase RNA containing modified nucleosides to cells allows for therapid and hypoimmunogenic or nonimmunogenic and RNA is a useful approachto telomere extension for diverse applications.

Methods

modRNA Template Generation and Synthesis.

The wild type (WT) human TERT open reading frame (ORF) used to generatethe DNA templates for modRNA synthesis is identical to the NCBI humanTERT transcript variant 1 reference sequence NM_198253.2, and wasgenerated by making the modification G516D to the ORF of thepBABE-neo-hTERT plasmid (Addgene plasmid 1774). Residue 516 is in theQFP motif of the N-terminal extension of TERT, a motif associated withmultimerization and RNA binding of TERT. The catalytically inactive TERT(CI TERT) mutant was generated from the WT TERT sequence by introducingthe mutation D712A. The WT and CI, TERT ORFs were inserted into the MCSof a starting plasmid containing the T7 promoter, the 5′ UTR of humanbeta globin (hBB), the MCS, the 3′ UTR of hBB, a 151 bp poly-A sequence,and a restriction site for linearization with class IIs enzyme followingthe poly-A sequence. The resulting intermediate plasmid was sequenced inat least quadruplicate to ensure fidelity, linearized, and transcribedto capped RNA using the RNA polymerase from the MEGAscript T7 Kit(Ambion, Austin, Tex.) and a custom nucleotide mix of canonical andnon-canonical nucleotides (TriLink BioTechnologies) in which the finalnucleotide concentrations per 40 ul IVT reaction were 7.5 mM for each ofadenosine-5′-triphosphate (ATP), 5-methylcytidine-5′-triphosphate (m5C),and pseudouridine-5′-triphosphate 0-10, 1.5 mM forguanosine-5′-triphosphate (GTP), and 6 mM for the cap analog (ARCA,NEB), or a molar ratio of ATP:m5C:T:GTP:ARCA of 1:1:1:0.2:0.8. Tofurther decrease potential immunogenicity of the mRNA related to the5′-3P-bearing fraction (˜20% of total) the IVT products were treatedwith phosphatase (Antarctic Phosphatase, NEB). The size and integrity ofthe modRNA products was verified using denaturing agarose gelelectrophoresis.

Cells and Cell Culture.

MRC-5 human fetal lung fibroblasts (ATCC CCL-171) were received fromATCC at passage 14, the current passage number of their distributioninventory. Since ATCC does not indicate the PD number, the PD valuescited herein refer to the number of PD after receipt of cells from ATCC,defined here as PD 0. To shorten their telomeres in preparation fortelomere extension experiments, cells were cultured using ATCCguidelines in DMEM with 10% FBS, in ambient oxygen and 5% CO₂. Telomereextension treatment began at approximately PD 40 after receipt of cellsfrom ATCC. At least 48 hours before the start of modRNA treatment cellswere transferred to 5% oxygen and DMEM medium containing 20% FBS andpenicillin-streptomycin. Cells were treated at least 24 hours afterplating and 24 hours before trypsinization.

modRNA Transfection.

Cells were transfected with 0.4 nM TERT modRNA and 2.0 nM TERC RNAunless otherwise specified, using Lipofectamine RNAiMax (Invitrogen), acationic lipid, in Opti-MEM Reduced Serum Media (Invitrogen) for 4-5hours, after which they were returned to normal medium.

Telomerase Activity Measurement.

24 hours after the start of transfection period, cells were washed withPBS, trypsinized, pelleted at 500 g for 5 min. and washed with PBS,repelleted, and then lysed following the instructions in the TRAPeze RTkit (Millipore). The reverse transcription and qPCR steps were carriedout in a Roche LightCycler 480 II and an ABI 7900 HT. Telomeraseactivity of samples was always compared to the reference standardprovided with the kit, as we found that fold-increase was prone to behighly variable, probably due to the low telomerase activity in MRC-5cells.

Immunocytochemistry.

24 hours after the start of transfection period, cells were washed withPBS, fixed in 2% paraformaldehyde for 20 minutes, washed three timeswith PBS, blocked in PBS containing 7.5% BSA and 0.1% Triton X-100 for 1hour, washed three times, incubated overnight at 4° C. on a rocker inanti-TERT antibody (ABCAM 32020 at 1:50 or Rockland 600-401-252S at1:500) in PBS with 5% BSA, washed three times, incubated for one hour insecondary antibody, washed two times then two more times on a shaker for3 minutes each time, then incubated in 0.1 ug/ml DAPI for 3 minutes, andwashed four times in PBS.

Flow Cytometry.

Cells treated with 0.5-1.5 ug/ml of TERT modRNA and controls weretrypsinized 22 h. after start of transfection, washed, fixed in 2%paraformaldehyde, permeabilized in 7.5% BSA with 0.1% Triton X-100 for20 min., washed three times in PBS, blocked in 7.4% BSA for 1 h,incubated in anti-TERT antibody (ABCAM 32020 at 1:50) in PBS with 5%BSA, washed three times, incubated in secondary antibody (1:200) in PBS,then washed three times and resuspended in FACS buffer and analyzed on aAccuri C6 Flow Cytometer (Becton Dickinson). Mean fluorescence intensitywas calculated as the average fluorescence of all cells in treated andcontrol samples. Data were analyzed using CFlow Plus software.

RT-PCR Total RNA was harvested using the RNeasy Mini kit (Qiagen) andconverted to cDNA using the High-Capacity RNA-to-cDNA Kit (Invitrogen).cDNA was amplified using PCR with the following primers:

hTERT-F: (SEQ ID NO: 1) 5′-GCCCTCAGACTTCAAGACCA 3′-hBB-R: (SEQ ID NO: 2)5′-AGGCAGAATCCAGATGCTCA GAPDH-F: (SEQ ID NO: 3) 5′-GTGGACCTGACCTGCCGTCTGAPDH-R: (SEQ ID NO: 4) 5′-GGAGGAGTGGGTGTCGCTGT

Western Blot.

Protein was harvested by washing cells once with PBS and then lysingcells in RIPA buffer. Protein was run on NuPAGE Novex Tris-Acetate Gels,transferred to PVDF membrane for 2 h. at 35 V, then hybridized toanti-alpha tubulin (Sigma at 1:10,000) and anti-TERT antibody (ABCAM32020 at 1:1000 or Rockland 600-401-252S at 1:500) and anti-overnight at4° C. Secondary detection was performed using infrared (680 nm and 800nm) antibodies (LI-COR) and the Odyssey imager (LI-COR). Images wereanalyzed using ImageJ.

Q-FISH.

Cells were incubated in 0.1 ug/ml colcemid for 4 hours before fixationand preparation of metaphase spreads following the procedure of Lansdorpet al. 41 Slides were then stained using the Telomere PNA FISH Kit/FITCkit (Dako, Denmark), substituting AlexaFluor 555-labeled telomere probe(Bio-Synthesis, USA) for the kit probe. Each cell was imaged on aDeltaVision (Applied Precision, Inc., Washington) microscope usingSoftWoRx software using either a 60× or 100× oil objective at 0.2 micronintervals over a range of 3 microns using a motorized stage, and customsoftware was used to identify the image in which each telomere spot wasin focus and integrate its intensity in that in-focus image. Temporalvariation in illumination intensity was compensated for using theDeltaVision photosensor which recorded the illumination intensity ofeach image, and variation in spatial intensity and CCD dark current andread error was compensated for by acquiring flat field images and darkfield images, respectively. At least 15 metaphase cells contributed toeach replicate and each sample was measured in at least duplicate overat least two separate experiments.

Growth Curves.

Cells were harvested and counted on a hemocytometer as an automatedcounter was unable to accurately count the cells when the populationbecame heterogeneous with respect to diameter as the population enteredreplicative senescence. Images were acquired to allow measurement ofcell diameters. PD were calculated as the log 2 of the ratio of cellnumbers at end and start of each culture period between passages.

Statistics.

Statistical analysis was performed using Microsoft Excel. Error barsrepresent the mean±s.e.m. Telomere lengths were compared using T-testswith P<0.05 (two-tailed) considered to be statistically significant.

Example 2. Effects of TERT modRNA Treatment on Human Endothelial Cells

The effects of TERT modRNA treatment is not limited to fibroblast cells.As shown in FIG. 9, human microvascular dermal endothelial cells(“HMDEC”) display typical doubling curves, with early onset ofsenescence at approximately passage #12 and with cells completelysenescent at passage #17. As shown in FIG. 10, treatment of these cellsat passage #18 with wild-type hTERT modRNA (right bar in each series)results in the reversal of senescence, whereas control treatment (leftbar in each series), or treatment with mutant hTERT modRNA (middle barin each series), had no effect on cellular senescence.

FIG. 11 demonstrates the effects of various treatments on the growth ofHMDECs. Specifically, cells treated three times with wild-type TERTmodRNA (WT) between passage #10 and passage #11 continued to grow,whereas untreated cells (UT), cells treated with carrier only (CO), andcells treated with a catalytically-inactive TERT (CI) stopped doubling.

FIG. 12 shows the results of β-galactosidase (bGAL) staining of HMDECsat passage #14 following treatment with WT and CI modRNAs. This stainingmeasures β-galactosidase activity at pH 6, which is a property ofsenescent cells that is not found in presenescent, quiescent, orimmortal cells. See Dimri et al. (1995) Proc. Nat'l Acad. Sci. USA92:9363-7.

Methods

The HMDECs Cumulative Population Doubling Curve was built using a12-well plate format. Approximately 10⁵ of HMDECs were at each timepoint replated in triplicates and cell counting was done on day 4 afterthe previous replating. As shown in FIG. 9, passage #12 was determinedas the early onset point of HMDECs senescence.

As shown in FIG. 10, approximately 10⁵ HMDECs were transfected twiceevery other day with 0.75 mg of modRNAs encoding hTERT-CI, and -WT inpresent of Carrier Only (CO, RNiMAX) control. Cell counting was done onPassages #19, 20, and 21 each time on day 5 after replating.

For the experiment shown in FIG. 11, in the interval between passage #10and #11 (and, as noted above, passage #12 corresponds to the early onsetof HMDEC senescence), approximately 5×10⁵ HMDECs per 75 cm³ flask werecultivated on EBM-2 medium supported with EGM-2 MV supplement (both fromLonza, Walkersville, Md. USA, catalogue nos. CC-3156 and CC-4176,respectively). Microvascular cells were transfected three times everyother day with 5 μg of modRNAs encoding hTERT-WT (WT) and hTERT-CI (CI)in the presence of untreated (UT) and carrier only (CO, RNAiMAX) controlflasks. At each time point, 5×10⁵ cells were plated and, starting frompassage #11, a cell count via hemocytometer was done on 5 days afterreplating. For each time point, each experimental condition wasperformed in triplicate.

For the experiments shown in FIG. 12, approximately 10⁵ of each NT,hTERT-CI and hTERT-WT HMDECs of passage #14 were treated as described inFIG. 10. The cells were then subjected to senescent cell analysis basedon a histochemical stain for β-galactosidase activity at pH 6 using aSenescence Cells Histochemical Staining Kit (Sigma, catalogue no.CS0030). The left side of the slide shows representative images ofnon-treated (NT) cells and cells treated with hTERT-CI (CI) or hTERT-WT(WT) modRNAs. All images were obtained under identical image-acquisitionconditions. Each experimental condition was performed in triplicate, andthe percent of cells positive for β-galactosidase activity (asrepresented in the chart) for each condition was calculated as anaverage of positive cells in three randomly captured areas.

Example 3. Further Characterization of the Effects of TERT modRNATreatment on Human Cells

In this example, the mRNA was transfected via a cationic lipid intoprimary human fibroblasts and myoblasts (FIG. 13A), cells known to havelimited proliferative capacity. Webster and Blau (1990) Somat Cell MolGenet. 16:557-565; Hayflick and Moorhead (1961) Exp Cell Res.25:585-621; Yakubov et al. (2010) Biochem Biophys Res Commun.394:189-193. Transfection efficiency was determined using flowcytometric single cell quantitation of fluorescence following deliveryof GFP mRNA, which showed that most cells (>90%) were transfected evenat relatively low concentrations of modified mRNA (0.1 μg/ml) (FIG. 13Band FIG. 16A-C). Treatment of cells with equal concentrations ofexogenous TERT mRNA or mRNA encoding a catalytically inactive (CI) formof TERT resulted in internalization of similar amounts of mRNA (FIG.16D), as measured by RT-qPCR 24 h after the first treatment. CI TERT hasa substitution mutation at one of the triad of metal-coordinatingaspartates at the catalytic site of the reverse transcriptase domain ofTERT. As a result, CI TERT cannot add nucleotides to telomeres, yetremains structurally intact, able to bind template DNA, and exhibitsstability comparable to wild type TERT in reticulocyte lysates. Wyatt(2009) Structure-Function Analysis of the Human Telomerase ReverseTranscriptase. Neither TERT nor CI TERT mRNA treatment affected levelsof endogenous TERT mRNA relative to untreated cells as measured byRT-qPCR (FIG. 16E). Transfection with 1 μg/ml of either TERT or CI TERTmRNA resulted in equivalent 50% increases (P<0.05 and <0.01,respectively) in the amount of TERT protein in fibroblasts (FIG. 13C,left panel). Wick et al. (1999) Gene. 232:97-106; Ahmed et al. (2008) JCell Sci. 121:1046-1053. The presence of endogenous TERT protein incells with little endogenous telomerase activity is consistent with therelative abundance of inactive splice variants of TERT in many celltypes and extensive post-translational inhibitory regulation of TERTactivity as previously reported by others. Yi et al. (2001) NucleicAcids Res. 29:4818-4825; Cifuentes-Rojas and Shippen (2011) Mutat Res.[published online ahead of print: Oct. 18, 2011];doi:10.1016/j.mrfmmm.2011.10.003. Treatment with increasing amounts ofTERT mRNA resulted in a dose-dependent increase of TERT proteinexpression as measured in single cell assays by flow cytometry (FIG.13C, right panel).

Telomerase Activity is Transiently Increased.

To test whether modified TERT mRNA delivery resulted in the generationof functional TERT protein, telomerase activity was quantified using agel-based TRAP assay. Telomerase activity was detected in fibroblastsand myoblasts at all doses of TERT mRNA tested (0.25, 0.5, 1.0, and 2.0μg/ml), and was not detected in untreated cells or cells treated witheither vehicle only or modified mRNA encoding CI TERT, even at thehighest dose of 2.0 μg/ml (FIG. 13D). Although TERT requires TERC toform a functional telomerase complex, delivery of TERT alone wassufficient to increase telomerase activity, consistent with previousfindings that TERT is often limiting as TERC RNA copy number is high inmany cell types lacking telomerase activity, including fibroblasts. Yiet al. (2001) Nucleic Acids Res. 29:4818-4825. A time course revealedthat telomerase activity peaked at 24 hours and returned to baselinelevels within 48 hours after a single transfection. This time frame isconsistent with previously reported half-lives of human TERT mRNA (2-4h), human β-globin mRNA (17-18 h: our exogenous TERT mRNA is flanked byβ-globin 5′ and 3′ UTRs), and telomerase activity in cells exposed to aninhibitor of protein synthesis (cell type dependent, but typically >24h). Kabnick and Housman (1988) Mol Cell Biol. 8:3244-3250; Holt et al.(1997) Proc Natl Acad Sci USA. 94:10687-10692; Xu et al. (1999) Br JCancer. 80:1156-1161.

Lengthening of Telomeres.

Telomere lengths in untreated fibroblasts declined over time (3 months)as expected (62) (FIG. 14A) and was quantified using two differentmethods. The monochrome multiplex qPCR method (MMqPCR) was used toassess length, and measurements were validated independently with a qPCRmethod performed by SpectraCell Laboratories, Inc. (correlationcoefficient 0.97, P<0.001). Delivery of TERT mRNA three times insuccession at 48-hour intervals to fibroblasts or myoblasts starting atpopulation doubling (PD) 25 and 6, respectively, extended telomeres by0.9±0.1 kb (22±3%), and 0.7±0.1 kb (12±2%), respectively (FIG. 14B,C).Treatment with vehicle only or CI TERT mRNA had no significant effect ontelomere length relative to untreated cells. The average rate oftelomere extension in fibroblasts was 135±15 bp/PD.

Cell Type-Dependent Increases in Proliferative Capacity.

To test the effect of modified TERT mRNA delivery and consequenttelomere extension on cell proliferative capacity, human fibroblastswere transfected either once, twice, or three times in succession.Treatments were delivered at 48-hour intervals. Untreated, vehicleonly-treated, and CI TERT mRNA-treated fibroblasts exhibited anequivalent plateau in cell number after approximately 50-60 PD, whereascells treated three times with TERT mRNA continued to proliferate for afinite additional 28±1.5 PD with an overall increase in cell number of2.7×10⁸ beyond untreated cells (FIG. 14D, left panel). The effect wasdose-dependent with each additional treatment conferring additional PD(FIG. 14D, right panel). The incremental increase in proliferativecapacity was greater with the first treatment than with the second orthird treatments. Human myoblasts treated three times in successionevery 48 hours gained 3.4±0.4 PD, equivalent to a 10-fold increase incell number compared to untreated or vehicle treated controls (FIG.14E). Such differences in PD between myoblasts and fibroblasts are notunexpected, as prior studies found similar limited effects of TERToverexpression to a few PD and showed that this limitation was due to ap16-mediated growth arrest in human myoblasts, in contrast tofibroblasts. Bodnar et al. (1998) Science. 279:349-352; Zhu et al.(2007) Aging Cell. 6:515-523. In both fibroblasts and myoblasts, vehicleonly or CI TERT mRNA had no effect on proliferative capacity compared tountreated controls. These data show that delivery of modified TERT mRNAis an effective method for increasing PD in culture. Importantly, all ofthe treated cells studied exhibited a significant increase in cellnumbers, but eventually reached a plateau in their growth curves,demonstrating absence of immortalization.

Transient Reduction in Markers of Senescence.

As the fibroblast populations stopped growing they exhibited markers ofsenescence including senescence-associated β-galactosidase (β-gal)staining and enlarged size (FIG. 15A-C). Cristofalo and Kritchevsky(1969) Med Exp Int J Exp Med. 19:313-320; Dimri et al. (1995) Proc NatlAcad Sci USA. 92:9363-9367; Cristofalo et al. (2004) Mech Ageing Dev.125:827-848; Lawless et al. (2010) Exp Gerontol. 45:772-778. Thesechanges were transiently reduced in fibroblasts treated with TERT mRNArelative to untreated cells and cells receiving CI TERT mRNA or vehicleonly. In accordance with findings by others, not all cells in thepopulations that had entered a growth plateau expressed β-galactosidaseat detectable levels. Lawless et al. (2010) Exp Gerontol. 45:772-778;Binet et al. (2009) Cancer Res. 69:9183-9191. However, TERTmRNA-transfected fibroblasts and myoblasts expressed β-galactosidase tothe same degree as the control cells of each type after the twopopulations reached a growth plateau. These data demonstrate that cellstreated with TERT mRNA eventually and predictably cease division andexpress markers of senescence, and are therefore unlikely to betransformed.

This example demonstrates that transient delivery of TERT mRNAcomprising modified nucleotides extends human telomeres and increasescell proliferative capacity without immortalizing the cells. The rate oftelomere extension in fibroblasts observed here of 135±15 bp/PD iscomparable to rates reported using viral methods, from 94 to >150 bp/PD(22, 69). Modified TERT mRNA extended telomeres in fibroblasts in a fewdays by 0.9±0.1 kb. Fibroblast telomere lengths have been reported toshorten over a human lifetime by approximately 1-2 kb on average.Allsopp et al. (1992) Proc Natl Acad Sci USA. 89:10114-10118. Thus,modified TERT mRNA is efficacious, yet transient and non-integrating,overcoming major limitations of constitutively expressed viral TERT mRNAdelivery.

Human cells of greatest interest are often limited in number, includingstem cells for use in experimentation or regenerative medicine. Thisproblem is currently being addressed by various methods includingsomatic nuclear transfer, viral methods for gene delivery, and the useof culture conditions that lessen the rate of telomere shortening. Le etal. (2013) Cell Stem Cell. [published online ahead of print: Nov. 19,2013]; doi:10.1016/j.stem.2013.11.005; Zimmermann and Martens (2008)Cell Tissue Res. 331:79-90; Mohsin et al. (2013) Circ Res.113:1169-1179. The modified TERT mRNA treatment described here providesan advantageous complement or alternative to these methods that isbrief, extends telomeres rapidly, and does not risk insertionalmutagenesis. The brevity of TERT mRNA treatment is particularlyattractive in that it can avert the loss of stem cell phenotype that canoccur over time in culture (Gilbert et al. (2010) Science.329:1078-1081) and shorten the post-reprogramming stage of iPSCgeneration during which telomeres extend (Wang et al. (2012) Cell Res.22:757-768). Such a method of extending telomeres has the potential toincrease the utility of diverse cell types for modeling diseases,screening for ameliorative drugs, and use in cell therapies.

A spectrum of effects on proliferative capacity was observed for thecell types tested, in agreement with previous studies demonstratingdifferent effects of TERT overexpression on myoblast and fibroblastproliferative capacity. Bodnar et al. (1998) Science. 279:349-352; Zhuet al. (2007) Aging Cell. 6:515-523. Moreover, the amount of telomereextension did not correlate with proliferative capacity. Thus, cellcontext determines the efficacy of TERT expression on proliferativecapacity and an understanding of the factors mediating this effect is ofinterest in overcoming this limitation. Factors that have beenimplicated in limiting myoblast proliferative capacity upon viral TERToverexpression include p16-mediated growth arrest, cell type and strain,and culture conditions. Zhu et al. (2007) Aging Cell. 6:515-523. Moregenerally, the effect may be mediated by non-telomeric DNA damage, age,and mitochondrial integrity. Sahin et al. (2011) Nature. 470:359-365;Mourkioti et al. (2013) Nat Cell Biol. 15:895-904; Lopez-Otin et al.(2013) Cell. 153:1194-1217. The absence of an increase in telomerelength or cell proliferative capacity in CI TERT mRNA-transfected cellsis consistent with the treatment acting through the catalytic site ofTERT by which nucleotides are added directly to telomeres. TERTmRNA-treated cell populations increased in number exponentially for aperiod of time and then eventually ceased expanding and exhibitedmarkers of senescence to a similar degree as untreated populations,consistent with the absence of immortalization.

The transient non-integrating nature of modified mRNA and finiteincrease in proliferative capacity observed here render it safer thancurrently used viral or DNA vectors. Further, the method extendstelomeres rapidly so that the treatment can be brief, after which theprotective telomere shortening mechanism remains intact. This method canbe used ex vivo to treat cell types that mediate certain conditions anddiseases, such as hematopoietic stem cells or progenitors in cases ofimmunosenescence or bone marrow failure. In addition, modified mRNA maybe delivered to certain tissues in vivo. Kormann et al. (2011) NatBiotechnol. 29:154-157. In summary, the rapid and safe method for rapidextension of telomeres described here leads to delayed senescence andincreased cell proliferative capacity without immortalizing human cells.

Methods

mRNA Template Generation and Synthesis.

To generate modified mRNA encoding GFP, TERT, and CI TERT, theirrespective open reading frames (ORFs) were inserted into the MCS of astarting plasmid containing the T7 promoter, the 5′ UTR of humanb-globin (HBB), the MCS, the 3′ UTR of HBB, a 151 bp poly-A sequence,and a restriction site for linearization with a class II enzymefollowing the poly-A sequence. The resulting intermediate plasmids weresequenced, linearized, and transcribed using the buffer and RNApolymerase from the MEGAscript T7 Kit (Ambion, Austin, Tex., USA), and acustom mix of canonical and non-canonical nucleotides (TriLinkBioTechnologies, San Diego, Calif., USA) in which the final nucleotideconcentrations per 40 μl WT reaction were 7.5 mM for each ofadenosine-5′-triphosphate (ATP), 5-methylcytidine-5′-triphosphate (m5C),and pseudouridine-5′-triphosphate (Ψ), 1.5 mM forguanosine-5′-triphosphate (GTP), and 6 mM for the cap analog (ARCA) (NewEngland Biolabs, Ipswitch, Mass., USA), or a molar ratio ofATP:m5C:Ψ:GTP:ARCA of 1:1:1:0.2:0.8. To further decrease potentialimmunogenicity of the mRNA related to the 5′-3P-bearing fraction, theIVT products were treated with Antarctic Phosphatase (New EnglandBiolabs). The size and integrity of the mRNA products were verifiedusing denaturing agarose gel electrophoresis. The wild type human TERTORF used to generate the DNA templates for mRNA synthesis is identicalto the NCBI human TERT transcript variant 1 (reference sequenceNM_198253.2). The ORF was generated from the pBABE-neo-hTERT plasmid(Counter et al. (1998) Proc Natl Acad Sci USA. 95:14723-14728) (plasmid1774, Addgene, Cambridge, Mass., USA). The pBABE-neo-hTERT plasmid had anon-silent mutation at residue 516 in the QFP motif of TERT, a motifassociated with multimerization and TERT interaction with TERC RNA, andthus to avoid the possibility of artifacts due to this mutation we madethe sequence identical to the NCBI reference sequence by correcting themutation with the change G516D. The CI TERT mutant was generated fromthe TERT sequence by introducing the mutation D712A.

Cell Culture and Treatment.

Human primary fetal lung MRCS fibroblasts were obtained from ATCC(Manassas, Va., USA) at passage 14. ATCC does not indicate the PDnumber, thus, our PD values cited herein refer to the number of PD afterreceipt of cells from ATCC. MRCS cells were cultured in DMEM with 20%FBS and penicillin-streptomycin. Human 30 year-old primary skeletalmuscle myoblasts (Lonza, Allendale, N.J., USA) were cultured in SkGM-2media (Lonza) according to the vendor's instructions. Populationdoublings were calculated as the base 2 log of the ratio between cellsharvested and cells plated at the previous passaging, and wereconsidered to be zero if fewer cells were harvested than plated. Cellswere transfected with modified TERT mRNA using Lipofectamine RNAiMax(Life Technologies, Grand Island, N.Y., USA) prepared in OptiMEM ReducedSerum Media (Life Technologies, Grand Island, N.Y., USA) and added tothe cells in a 1:5 v:v ratio with their normal media to achieve thefinal concentrations indicated herein.

Telomerase Activity Measurement.

Twenty-four hours after the start of the transfection period, cells wereharvested and lysed in CHAPS buffer. The TRAP assay was performed usinga modified version of the TRAPeze kit (EMD Millipore, Billerica, Mass.,USA), in which the primers and polymerase were added after, rather thanbefore, the step during which the artificial telomere substrate isextended. The PCR program was 94° C. 30 s/59° C. 30 s/72° C. 45 s for 30cycles, and the products were run on a 15% polyacrylamide gel in 0.5×TBEstained with SYBR Gold Nucleic Acid Gel Stain (Life Technologies, GrandIsland, N.Y., USA). The time course of telomerase activity was performedusing the TRAPeze RT kit (EMD Millipore, Billerica, Mass., USA).

Western Blot.

Protein was harvested by washing cells once with PBS and then lysingcells in RIPA buffer (Cell Signaling Technology, Danvers Mass., USA).Protein was run on NuPAGE Novex Tris-Acetate Gels (Life Technologies,Grand Island, N.Y., USA), transferred to PVDF membrane for 2 h at 35V,then hybridized to anti-α tubulin (Sigma, St. Louis, Mo., USA) at1:10,000 and anti-TERT antibody (ABCAM, Cambridge, Mass., USA, 32020 at1:1000; or Rockland Immunochemicals, Gilbertsville, Pa., USA,600-401-252S at 1:500) and incubated overnight at 4° C. Detection wasperformed using infrared (680 nm and 800 nm) antibodies (LI-COR,Lincoln, Nebr., USA) and the Odyssey imager (LI-COR). Total intensity ofeach band was quantified using ImageJ (NIH, Bethesda, Md., USA). Theintensity of each TERT band was normalized by its corresponding atubulin band.

Flow Cytometry.

Cells were harvested 24 h after transfection with the indicated doses(FIG. 16A-C) of TERT mRNA and stained with anti-TERT antibody (RocklandImmunochemicals, Gilbertsville, Pa., USA; 600-401-252S) at 1:500.

Telomere Length Measurement by SpectraCell Laboratories, Inc.

Genomic DNA was extracted using phenol chloroform and quantified usingthe Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, GrandIsland, N.Y., USA). Telomere length analysis was performed atSpectraCell Laboratories Inc. (Houston, Tex., USA) using a CLIAapproved, high throughput qPCR assay, essentially as described byCawthon et al. Cawthon (2002) Nucleic Acids Res. 30(10):e47; Cawthon(2009) Nucleic Acids Res. 37(3):e21. The assay determines a relativetelomere length by measuring the factor by which the sample differs froma reference DNA sample in its ratio of telomere repeat copy number tosinge gene (36B4) copy number. This ratio (T/S ratio) is thought to beproportional to the average telomere length. All samples were run in atleast duplicate with at least one negative control and two positivecontrols of two different known telomere lengths (high and low) and anaverage variance of up to 8% was seen. The results were reported as atelomere score equivalent to the average telomere length in kb.

Telomere Length Measurement by MMqPCR.

Telomere length was measured using a modified version of the MMqPCRprotocol developed by Cawthon (Cawthon (2009) Nucleic Acids Res.37(3):e21) with the following changes: Additional PCR pre-amplificationcycles were added to make the telomere product amplify earlier, wideningthe gap between telomere and single-copy gene signals; a mixture of twoTaq polymerases was experimentally determined to result in better PCRreaction efficiencies than each on its own; reducing the SYBR Greenconcentration from 0.75× to 0.5× resulted in earlier signal. Genomic DNAwas harvested from cells using the PureGene kit (Qiagen Germantown, Md.,USA) with RNase digestion, quantified using a NanoDrop 2000(ThermoFisher Scientific, Waltham, Mass., USA), and 10-40 ng was usedper 15 μl qPCR reaction performed in quadruplicate using a LightCycler480 PCR System (Roche, Basel, Switzerland). A serial dilution ofreference DNA spanning five points from 100 ng/μl to 1.23 ng/μl wasincluded in each assay to generate a standard curve required for sampleDNA quantification. The final concentrations of reagents in each 15 μlPCR reaction were: 20 mM Tris-HCl pH 8.4, 50 mM KCl, 3 mM MgCl2, 0.2 mMeach dNTP, 1 mM DTT, 1 M betaine (Affymetrix, Santa Clara, Calif., USA),0.5× SYBR Green I (Life Technologies, Grand Island, N.Y., USA), 0.1875 UPlatinum Taq (Life Technologies, Grand Island, N.Y., USA), 0.0625×Titanium Taq (Clontech), and 900 nM each primer (telg, telc, hbgu, andhbgd primer sequences specified in Cawthon (2009) Nucleic Acids Res.37(3):e21. The thermal cycling program was: 2 minutes at 95° C.;followed by 6 cycles of 15 s at 95° C., 15 s at 49° C.; followed by 40cycles of 15 s at 95° C., 10 s at 62° C., 15 s at 72° C. with signalacquisition, 15 s at 84° C., and 10 s at 88° C. with signal acquisition.The Roche LightCycler 480 software was used to generate standard curvesand calculate the DNA concentrations of telomere and single-copy genesfor each sample. T/S ratios were calculated for each sample replicate,and the result averaged to yield the sample T/S ratio which wascalibrated using blinded replicate samples of reference cells sent toSpectraCell as described above. The independently obtained relativevalues of T/S ratios measured using MMqPCR and by SpectraCell for thesame samples were highly consistent (correlation coefficient=0.97,P<0.001).

Reverse Transcription qPCR.

Primers were designed using Primer3 (Untergasser et al. (2012) NucleicAcids Res. 40:e115) and are listed in Table 9 except where otherwisenoted. Twenty-four hours after start of treatment, cells were washedthree times with PBS before harvesting in Buffer RLT (Qiagen,Germantown, Md., USA). RNA was converted to cDNA using High CapacityRNA-to-cDNA Master Mix (Life Technologies, Grand Island, N.Y., USA).Endogenous TERT mRNA was amplified using a forward primer in the openreading frame of TERT and a reverse primer in the 3′ UTR of endogenousTERT mRNA. Exogenous TERT mRNA was amplified using a forward primer inthe open reading frame of TERT mRNA and a reverse primer in the 3′ UTRof HBB present in our exogenous TERT and CI TERT mRNA, but not inendogenous TERT mRNA. Relative levels were calculated using the Pfafflmethod. Reference genes were RPL37A (using primers specified in Greberet al. (2011) EMBO J. 30:4874-4884) and GAPDH, neither of whichexhibited a significant change in Ct value in control or treated cells.

TABLE 9 Primer sequences.   Product Forward primer length Target (5′-3′)Reverse primer (5′-3′) (bp) Exogenous hTERT (NM_198253.2) 3′ UTR of HBB162 TERT GTCACCTACGTGCCACTCCT AGCAAGAAAGCGAGCCAAT (SEQ ID NO: 5)(SEQ ID NO: 6) Endogenous hTERT (NM_198253.2) 3′UTR of hTERT (NM_198253.2)  74 TERT GCCCTCAGACTTCAAGACCAGCTGCTGGTGTCTGCTCTC (SEQ ID NO: 7) (SEQ ID NO: 8) GAPDHCAATGACCCCTTCATTGACC TTGATTTTGGAGGGATCTCG 159 (SEQ ID NO: 9)(SEQ ID NO: 10)

Senescence-associated β-galactosidase staining and cell size scoring.β-gal staining was performed using the Senescence β-GalactosidaseStaining Kit (Cell Signaling Technology, Danvers Mass., USA). At least50 cells per population were scored in duplicate. Cell diameter wasscored manually after trypsinization on a hemocytometer grid. Cristofaloand Kritchevsky (1969) Med Exp Int J Exp Med. 19:313-320.

Statistics.

Student's T-tests and Pearson correlation coefficient calculations wereperformed using Microsoft Excel. Error bars represent the mean±s.e.m.

Example 4. Delivery of TERT modRNA to Cells by Electroporation

The TERT modRNA compounds of the invention may also be delivered tocells by electroporation, as illustrated in FIGS. 18-20, usingelectroporation parameters, including the concentration of TERT modRNA,voltage wave form, and electrode geometry appropriate for achievingoptimal transfection efficiency and viability in a given cell type. FIG.21 shows the dependence of telomerase activity of the dose of TERTmodRNA delivered by electroporation.

Example 5. Delivery of a modRNA to Human Blood Cells

CD8+ T-cells were transfected with modRNA as follows. The buffy layerfrom whole human blood centrifuged on Lymphoprep (Axis-Shield) densitygradient medium to obtain mononuclear cells which were washed twice andthen depleted of non-CD8+ leukocytes using the Dynabeads Untouched HumanCD8 T Cells Kit (Life Technologies). The CD8+ cells were stimulatedusing Dynabeads Human T-Activator CD3.CD28 (Life Technologies) andcultured for 4 days using OpTimizer T-Cell media supplemented with30,000 U/ml IL-2. The T-cells were then transfected with modRNA encodingnuclear GFP and TERT at a concentration of 50 μg/ml in a volume of 20 μlNucleofector P3 solution containing Supplement 1. Cells were assayed forfluorescence and viability 24 hours after transfection. Results of thetransfection are shown in FIG. 22. The bright fluorescence demonstrateshigh copy number, with transfection efficiency over 90% with highviability.

Example 6. Expression of Telomerase in Human Keratinocytes Using aTelomerase modRNA

As shown in FIG. 23, electroporation of human keratinocytes with TERTmodRNA results in the expression of telomerase activity in the cells.Human primary keratinocytes (Lonza) were suspended at a density of 3×10⁷cells/ml in OptiMEM media (Life Technologies) containing 50 μg/ml ofmodRNA encoding either catalytically inactive (CI) TERT or wild typeTERT. 10 μl of the cell suspension was placed in a 1 mm gap cuvette andelectroporated using a Gene Pulser (BioRad) using 200 V, 1000 Ohms, and25 microfarads. The cells were immediately returned to KGM-2 media(Lonza) and incubated for 24 hours before being harvested formeasurement of telomerase activity using the gel-based TRAPeze assay(Millipore). Each 25 μl TRAP reaction was performed using 1 μg of totalprotein, with duplicate samples heated at 85° C. for 10 minutes toinactivate telomerase. Untreated, electroporation only, and CI TERTsamples served as negative controls, and 293T cells and TSR8 served aspositive controls.

Example 6. Expression of a modRNA-Encoded Protein by Delivery In Vivo

modRNA delivered in vivo can result in the expression of a functionalprotein encoded by the modRNA. Kormann et al. (2012) NatureBiotechnology 29:154-157; Karikó et al. (2012) Molecular Therapy20:948-93. This has been confirmed here by complexing 2 μg of modRNAencoding luciferase with a cationic lipid vehicle (TransIT) andinjecting intravenously in 50 μl of OptiMEM (Life Technologies) in arodent tail. The spleen was harvested and treated with luciferin andassayed for luciferase activity using an IVIS bioluminescence imager(Perkin-Elmer). As shown in FIG. 24, luciferase activity was detected atthe injection site and in the spleen (arrows).

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in theirentireties as if each had been individually and specificallyincorporated by reference herein.

While specific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will become apparent tothose skilled in the art upon review of the specification. The scope ofthe invention should, therefore, be determined by reference to theappended claims, along with their full scope of equivalents.

What is claimed is:
 1. A method of extending telomeres, comprising thestep of: administering a compound to an animal cell wherein saidadministering is in vitro, wherein the compound comprises a syntheticribonucleic acid comprising at least one modified nucleoside and codingfor a telomerase reverse transcriptase, wherein the telomerase reversetranscriptase is expressed transiently in the cell, and wherein at leastone telomere is extended within the cell.
 2. The method of claim 1,wherein the cell has at least one shortened telomere prior to theadministering step.
 3. The method of claim 1, wherein the cell is fromor in a subject suffering from or at risk of an age-related illness, anage-related condition, or an age-related decline in function orappearance.
 4. The method of claim 1, wherein the cell is from or in asubject suffering from or at risk of cancer, heart disease, stroke,diabetes, diabetic ulcers, Alzheimer's disease, osteoporosis, a declinein physical ability or appearance, physical trauma or chronic physicalstress, psychological trauma or chronic psychological stress, reducedimmune function, immunosenescence, or macular degeneration.
 5. Themethod of claim 1, wherein the cell is a somatic cell of endodermal,mesodermal, or ectodermal lineage, or a germ line or embryonic cell. 6.The method of claim 1, wherein the cell is an induced pluripotent stemcell or a cell used to produce an induced pluripotent stem cell.
 7. Themethod of claim 1, wherein the cell is a transdifferentiated cell or acell used to produce a transdifferentiated cell.
 8. The method of claim1, wherein the cell is an isolated cell, and the administering steplasts no longer than 48 hours.
 9. The method of claim 1, wherein thecell is an isolated cell, and the administering step lasts at least 2hours.
 10. The method of claim 1, wherein the cell is an isolated cell,and the administering step is performed no more than four times.
 11. Themethod of claim 1, wherein the cell is an isolated cell, and theadministering step is performed at least two times.
 12. The method ofclaim 1, wherein the cell is an isolated cell, and the method furthercomprises the step of measuring telomerase activity in the cell.
 13. Themethod of claim 1, wherein the administering step increases telomeraseactivity in the cell.
 14. The method of claim 13, wherein the telomeraseactivity is transiently increased by at least 5%.
 15. The method ofclaim 13, wherein the half-life of increased telomerase activity is nolonger than 48 hours.
 16. The method of claim 13, wherein the half-lifeof increased telomerase activity is at least 2 hours.
 17. The method ofclaim 1, wherein the method further comprises the step of measuringtelomere length in the cell.
 18. The method of claim 1, wherein theadministering step increases average telomere length in the cell. 19.The method of claim 18, wherein average telomere length in the cell isincreased by at least 0.1 kb.
 20. The method of claim 1, wherein thecell is an isolated cell, and the method further comprises the step ofmeasuring population doubling capacity in the cell.
 21. The method ofclaim 1, wherein the administering step increases population doublingcapacity in the cell.
 22. The method of claim 21, wherein the populationdoubling capacity increases by at least one population doubling.
 23. Themethod of claim 1, wherein the cell is from or in a mammalian subject.24. The method of claim 23, wherein the cell is from or in a humansubject.
 25. The method of claim 1, wherein the cell is an isolatedcell.
 26. The method of claim 1, wherein the administering stepcomprises electroporation.
 27. The method of claim 1, wherein the atleast one telomere is transiently extended within the cell.
 28. Themethod of claim 1, wherein the compound is not administeredcontinuously.
 29. The method of claim 1, wherein said isolated cell ispart of a cell culture, an isolated tissue culture or an isolated organ.